US008586621B2

(12) Ulllted States Patent (10) Patent N0.: US 8,586,621 B2 Zeligs (45) Date of Patent: Nov. 19, 2013

(54) ANTI-PARASITIC METHODS AND 2003/0211165 A1 11/2003 Vogel

COMPOSITIONS UTILIZING ZSSZSZZQSZZ 21 155883 ghesv-lmihloup1 e a. DHNDOLYLMETHANE-RELATED INDOLES 2004/0022760 A1* 2/2004 McKenna et al...... 424/85.1 _ _ 2004/0142000 A1* 7/2004 Suga et al...... 424/195.15 (76) Inventor: MlchaelA- ZellgS, Boulder, CO (US) 2005/0158347 A1* 7/2005 Tarleton et al. 424/2691 2006/0100264 A1* 5/2006 Bjeldanes et al...... 514/414 ( * ) Notice: Subject to any disclaimer, the term of this 2006/0229355 A1 10/2006 Bjeldanes et al. patent is extended or adjusted under 35 Zoos/0145418 A1 6/2008 Zehgs et al' U.S.C. 154(b) by 257 days. FOREIGN PATENT DOCUMENTS (21) Appl. No.1 12/447,307 EP 0566226 “V1993 _ W0 WO 96/30347 3/1996 (22) PCT Flledi Oct- 26, 2007 W0 WO 96/33980 10/1996 W0 WO 97/02266 1/1997 (86) PCT No.1 PCT/US2007/022649 W0 WO 97/30034 8/1997 W0 WO 97/38983 10/1997 § 371 (0X1), W0 W0 99/55683 11/1999 (2), (4) Date: Apr. 27, 2009 W0 W0 00/02857 1/2000 W0 WO 00/31048 6/2000 . W0 WO 01/20990 3/2001 (87) PCT Pub. No.. WO2008/057253 W0 W0 02/092575 110002 PCT pub Date; May 15, 2008 W0 WO 2004/071425 5/2004 W0 WO 2005/107747 11/2005 . . . W0 WO 2006/047716 5/2006 (65) Prior Publication Data W0 W0 2006/053l60 * 50006 US 2010/0055201A1 Mar. 4, 2010 OTHER PUBLICATIONS

Related U.S.Application Data ______Rath et al. “Pharmacokmetrc study of Artemrsrnrn after oral rntake of (60) PrOVlslOnal apphcanon NO- 60/854,830, ?led on Oct atraditional preparation ofArtemisia annua L. (annual WormWood),” 27, 2006- Am. J. Trop. Med. Hyg. 2004 vol. 70, N0. 2, pp. 128-132.* 51 I Cl AggarWal & IchikaWa, 2005, “Molecular targets and anticancer ( ) nt' ' potential of indole-3-carbinol and its derivatives,” Cell Cycle A61K 31/40 (2006.01) 4(9):1201_15‘ A61K31/405 (200601) Akkar et al., 2003, “Formulation of intravenous carbamazepine emulsions by SolEmuls technology,” Eur J. Pharm Biopharm. ' 551305-12. A61K 9/00 (200601) Baugh et al., Jan. 23, 1998, “Treatment of cervical dysplasia With (52) U-s- Cl- indole-3-carbinol” in The Ray A. Barlow Scienti?c Symposium, USPC ------~- 514/415; 514/414; 514/420; 424/451; Shreveport : The Center for Excellence in Cancer Research, Treat 424/464; 424/400 ment and Education, Louisiana State University Medical Center, (58) Field of Classi?cation Search Shreveport (LA), p. 3. USPC ...... 514/414, 415, 420; 424/451, 464, 400 . See application ?le for complete search history. (Commued)

(56) References Cited Primary Examiner * Shengjun Wang U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm * Jones Day 5,635,215 A 6/1997 Boschettiet al. 5,718,921 A 2/1998 MahtioWitZ etal. 5,830,887 A 11/1998 Kelly (57) ABSTRACT 5,895,787 A 4/1999 Arffmann et al. 5,948,808 A 9/1999 Safe The present invention includes methods and compositions for 6,086,915 A 7/2000 Zeligs et al. the treatment and prevention of protoZoal parasitic infections 6,399,645 B1 6/2002 Bell et al. utilizing Diindolylmethane-related indoles. Additive and 6,477,229 B1 11/2002 Grosser 6,534,085 B1* 3/2003 Zeligs ...... 424/451 synergistic interaction of Diindolylmethane-related indoles 6,583,167 B2 6/2003 Palmer et al. With other knoWn anti-parasitic and pro-apoptotic agents is 6,613,792 B1 9/2003 Ellenberger et al. believed to permit more effective therapy and prevention of 6,656,963 B2 12/2003 Firestone et al. protoZoal parasitic infections. The methods and compositions 6,689,387 B1 2/2004 Zeligs 6,800,655 B2 10/2004 Jong et al. described provide neW treatment of protoZoal parasitic dis 7,348,352 B2 3/2008 Zeligs eases of mammals andbirds including , leishmaniasis, 7,384,971 B2 6/2008 Zeligs trypanosomiasis, trichomoniasis, neosporosis and coccidi 7,384,972 B2 6/2008 Zeligs 7,709,520 B2 5/2010 Safe os1s. 7,989,486 B2 8/2011 Zeligs et al. 8,080,577 B2 12/2011 Zeligs et al. 2002/0147155 A1 10/2002 Fosteretal. 53 Claims, N0 Drawings US 8,586,621 B2 Page 2

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Leong et al., 2004, “Potent ligand-independent estrogen receptor Bonnesen et al., 2001, “Dietary indoles and isothiocyanates that are activation by 3,3‘-diindolylmethane is mediated by cross talk generated from cruciferous vegetables can both induce apoptosis and between the protein kinase A and mitogen-activated protein kinase confer protection against DNA damage in human colon cell lines,” signaling pathways,” Mol Endocrinol. 18:291-302. Cancer Research, American Association for Cancer Research Liu et al., 1994, “Indolo[3,2-b]carbozole: a dietary-derived factor 61:6120-6130. that exhibits both antiestrogenic and estrogenic activity,” J. Natl. Brad?eld et al., 1987, “High-performance liquid chromatographic Cancer Inst. 86:1758-1765. Loria et al., 1990, “Immune response facilitation and resistance to analysis of anticarcinogenic indoles in Brassica oleracea,” J Agric virus and bacterial infectionis with dehydroepiandrosterone Food Chem 35:46-49. (DHEA),” Biologic Role of Dehydroepiandrosterone, pp. 107-130. Brad?eld et al., 1987, “Structure- Activity relationships of dietary Loub et al., 1975, “Aryl hydrocarbon hydroxylase induction in rat indoles: a proposed mechanism of action as modi?ers of xenobiotic tissues by naturally occurring indoles of cruciferous plants,” J. Natl. metabolism,” J Toxicol Environ Health 21:31 1-23. Cancer Inst. 54:985-988. Bradlow et al., 1999, “Multifunctional aspects of the action of indole Michnovicz et al., 1997, “Changes in levels of urinary estrogen 3-carbinol as an anti-tumor agent,” Annals of NewYork Academy of metabolites after oral indole-3 -carbinol treatment in humans”, J Natl Sciences 889: 204-213. Cancer Inst 89:718-23. Bradlow et al., 1996, “Z-hydroxyestrone: the ‘good’ estrogen,” J Mor?n R et al., 1994, “Pregnenolone and dehydroepiandrosterone as Endocrin 150:S259-S265. precursors of native 7-hydroxylated metabolites which increase the Brandi et al., 2003, “A new indole-3-carbinol tetrameric derivative immune response in mice,” J of Steroid Biochemistry and Molecular inhibits cyclin-dependent kinase 6 expression, and induces GI cell Biology, Elsevier Science Ltd., Oxford, GB, vol. 50(1/2) Jul. 1994, cycle arrest in both estrogen-dependent and estrogen-independent pp. 91-100. breast cancer cell lines,” Cancer Res. 63(14):4028-36. Muzandu et al., 2005, “Lycopene and beta-carotene ameliorate Calabro et al, 2005, “Inhibition of Tumor-Necrosis-Factor-or Induced catechol estrogen-mediated DNA damage,” Jpn J Vet Res. 52: 173-84. Endothelial Cell Activation by a New Class of PPAR-y: Agonists an Rahman et al., 2005, “Inhibition of Nuclear Translocation of Nuclear in vitro Study Showing Receptor-Independent Effects,” J Vasc Factor-kB Breast Cancer Cells Contributes to 3,3‘Diindolylmethane Induced Apoptosis in,” Cancer Res. 65:364-371. Res;42: 509-516. Riby et al., 2000, “Ligand-independent activation of estrogen recep Chang et al., 1999, “Cytostatic and antiestrogenic effects of tor function by 3,3‘-diindolylmethane in human breast cancer cells,” 2-(Indo1-3-ylmethyl)-3,3‘-diindolylmethane, a major in vivo product Biochem. Pharmacol. 60:167-177. of dietary indole-3 -carbinol,” Biochem. Pharmacol. 58:825-834. Ritter et al., 2001, “Oxidations of 17beta-estradiol and estrone and Chen et al., 1998, “Aryl hydrocarbon receptor-mediated their interconversions catalyzed by liver, mammary gland and mam antiestrogenic and antitumorigenic activity of dindoylmethane,” mary tumor after acute and chronic treatment of rats with indole-3 Carcinogenesis 19: 163 1-1639. carbinol or beta-naphtho?avine,” Can. J. Physiol. Pharmacol. Chen et al., 2001, “Indole-3-carbinol and diindolylmethane induce 79(6):519-32. apoptosis of human cervical cancer cells and in murine HPV16 Rogan, 2006, “The natural chemopreventive compound indole-3 transgenic preneoplastic cervical epithelium,” J Nutr. 131:3294-302. carbinol: state of the science” In Vivo. 20(2):221-8. Review. Dashwood, 1998, “Indole-3-carbinol: anticarcinogen or tumor pro Rosen et al., 1998, “Preliminary results of the use of indole-3 moter in brassica vegetables?” Chem Biol. Interact. 110(1-2):1-5. carbinol for recurrent respiratory papillomato sis,” Otolaryngol Head de Kruifet al., 1991,“. Structure elucidation of acid reaction products Neck Surg118:810-5. of indole-3 -carbinol: detection in vivo and enzyme induction in Roy et al, 2008,“An insight into the mechanism of inhibition of vitro,” Chem Biol Interact. 80:803-15. unusual bi-subunittopoisomerase I from Leishmania donovani by Del Cacho, 2004, “Expression of anti-apoptotic factors in cells para 3,3-di-indolylmethane, a novel DNA topoisomerase I poison with a sitized by second-generation schizonts of Eimeria tenella and strong binding af?nity to the enzyme,” Biochem. J. 409:611-622. Eimeria necatrix,” Vetina Parasitology 125:287-300. Roy et al., 2008. “Mitochondria-Dependent Reactive Oxygen Spe Del Prete, 1998, “The Concept of Type-1 and Type-2 Helper T Cells cies-Mediated Programmed Cell Death Induced by 3,3 and Their Cytokines in Humans,” Int Rev Immunol. 16(3-4): 427-55. Diindolylmethane through Inhibition of FOF1-ATP Synthase in Uni Exon et al., 2000, “Dietary indole-3-carbinol alters immune func cellular Protozoan Parasite Leishmania donovani,” Mol Pharmacol. tions in rats,” J. Toxicol. Environ. Health A. 59(4):271-9. 74:1292-1307. Gao et al., 2002, “Endocrine disruption by indole-3 -carbinol and Saudek et al., 1989, “A preliminary trial of the programmable tamoxifen: blockage of ovulation,” Toxicol Appl Pharmacol. implantable medication system for insulin delivery,” New Engl. J. 183 : 179-88. Med. 321:574-79. Gillner et al., 1985, “Interactions of indoles with speci?c binding Sefton, 1987, “Implantable pumps,” CRC Crit Ref., Biomed Eng. sites for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver,” Mol 14:201. Pharmacol 28:357-363. Sepkovic et al., 2001, “Quantitative Determination of 3,3‘ Hardvian,et al., “Goodman & Gilman’s The Pharmacological Basis Diindolyrnethane in the urine of individuals receiving indole-3 ofTherapeutics” (9th ed, 1996), pp. 51 and 57-58. carbinol,” Nutr Cancer. 41( l-2):57-63. US 8,586,621 B2 Page 3

(56) References Cited Tse et al., 1987, “Disposition of alpha-[(dimethylamino)methyl]-2 (3 -ethyl-5-methyl-4-isoxaZolyl)-1H-indole-3-methanol (59-801), a OTHER PUBLICATIONS hypoglycaemic agent in rats, dogs and monkeys,” Xenobiotica 17(6):741-9. Shilling et al., 2001, “3,3‘-diindolylmethane, a major condensation Woodburn et al, 1999, “The epidermal growth factor receptor and its product of indole-3-carbinol, is a potent estrogen in the rainbow inhibition in cancer,” Pharmacol. Ther. 82:241-250. trout,” Toxicology and Applied Pharmacology 170: 191-200. Yuan F et al., Jan. 9-15, 1999, “Prevention of Papillomavirus initiated cancer by the phytochemical Indole-3 -Carbinol,” Proceedings of the Smaill et al., 1999, “Tyrosine kinase inhibitors. 15. 17th International Papillomavirus Conference, p. 73. 4-(Phenylamino) and 4-(phenylamino)pyrido[d] Yuhas et al, 1990. “Inhibition of tumor necrosis factor-induced cell pyrimidine acrylamides as irreversible inhibitors of the ATP binding killing by tryptophan and indole” Eur. Cytokine Net. 1: 35-40. site of the epidermal growth factor receptor,” J Med Chem. 42: 1803 Zeligs et al., 2002, “Absorption-enhanced 3,3-dindolylinethane: 1815 human use in HPV-related, benign and pre-cancerous conditions,” Stewart et al., 2004, “Resveratrol antagonizes EGFR-dependent Proceedings of the Annual Meeting of the American Association for Erk1/2 activation in human androgen-independent prostate cancer Cancer Research, NewYork, NY vol. 43. Mar. 2002, p. 664, Abstract 3 198. cells with associated isoZyme-selective PKC alpha inhibition,” Zeligs, 1998, “Diet and Estrogen Status: The Cruciferous Connec Invest. New Drugs 22: 107-117. tion,” J Med Food 1:67-82. Stresser et al., 1995, “Mechanisms of tumor modulation by indole International Search Report, dated Sep. 22, 2008 of PCT/US2007/ 3-carbinol: disposition and excretion in male ?sher 344 rats,” Drug 022649 now published as WO2008/057253. Metabolism and Disposition 23:965-975. Written Opinion, dated Sep. 22, 2008 of PCT/US2007/022649 now Stresser et al., 1995, “The anticarcinogen 3,3‘-Diindolyl-methane is published as WO2008/057253. an inhibitor of cytochrome P-450,” J. Biochem. Toxicol. 10(4): 191 201. * cited by examiner US 8,586,621 B2 1 2 ANTI-PARASITIC METHODS AND metabolism requires novel pharmacologic approaches to ?nd COMPOSITIONS UTILIZING therapeutic agents selective for eliminating protoZoal organ DIINDOLYLMETHANE-RELATED INDOLES isms living Within an animal host. Unlike bacteria and viruses, protoZoa may assume different sexual forms and This application is the National Stage of International differentiate into a variety of maturational stages in various Application No. PCT/US2007/022649, ?led on Oct. 26, organs, presenting unique challenges for recognition by the 2007, Which claims the bene?t of US. Provisional Applica host immune system. As genetically more complex organ tion No. 60/854,830, ?led on Oct. 27, 2006. isms than bacteria and viruses, protoZoa differentiate into forms Which resist killing by knoWn microbicides active l. FIELD OF THE INVENTION against bacteria and viruses (Weir et al., 2002, Appl Environ Microbiol. 68(5):2576-9). Development of effective antipro The present invention relates to methods and compositions toZoal therapeutics and stimulation of host immune responses for the treatment and prevention of parasitic infections, pref against protoZoal parasites therefore requires approaches dif erably protoZoal parasitic infections, utilizing Diindolyl ferent from those utiliZed in developing antibacterial, antivi methane-related indoles. Administration of Diindolyl ral, and general immune potentiating agents. methane-related indoles, alone or in combination With knoWn The primary protoZoal parasites causing disease in man agents, provides a method to selectively pro include hemo?agellates of the class Trypanosomatidea, caus mote apoptosis of parasite infected host cells and of extra ing Leishmaniasis and Trypanosomiasis, and parasites of the cellular protoZoal parasites. Direct pro-apoptotic activity and phylumApicomplexa, class Coccidea, causing malaria, toxo interaction of Diindolylmethane-related indoles With other 20 plasmosis, cryptosporidiosis, and bebesiosis. Species of Coc knoWn anti-parasitic and pro-apoptotic agents is believed to cidea can infect humans, domestic animals and livestock, permit more effective chemotherapy and to be complemen including poultry, lambs, calves, piglets, and rabbits. Proto tary to co-administered anti -protoZoal vaccines. The methods Zoal parasitic diseases related to malaria include disease and compositions of the present invention provide neW thera caused by parasites of the species Neospora. Neospora infec peutic options in treating the major protoZoal parasitic dis 25 tions occur in dogs, cattle, sheep, goats and horses. eases of mammals and birds including malaria, leishmaniasis, The majority of populations in developing countries are trypanosomiasis, neosporosis and coccidiosis. noW at high risk of various protoZoal infections including malaria, leishmaniasis, and trypanosomiasis. Together these 2. BACKGROUND OF THE INVENTION protoZoal diseases cause millions of preventable deaths every 30 year. No preventive or therapeutic vaccines are yet available 2.1 ProtoZoal Related Disease for these parasitic diseases. The market for drugs against such diseases is limited by poverty and the emergence of resistance ProtoZoal parasites are single-celled organisms Which live to existing single agent chemotherapy. As used herein, che during some or all stages of their life cycle Within organs, motherapy refers to the use of chemical substances to treat tissues and cells of multicellular, metaZoan animals. As para 35 disease. Due to the lack of protective immunity folloWing sites, they obtain nutrients either from the host organism’s infection, With or Without chemotherapy, reinfection is a food supply or from its cells and tissues. As eukaryotic uni common phenomenon. Innovative and cost effective neW cellular organisms, the protoZoal parasites are able to live drugs and combination therapies using neW and old drug both Within animal cells and as free living extra-cellular para products are urgently needed. The development of broad sites residing in the blood, lymph tissue or Within the intesti 40 spectrum anti-parasitic agents able to be used in combination nal lumen. With existing chemotherapeutics is preferable to reduce the As agents of infection, the protoZoal parasites are funda emergence of neW resistance. An ideal anti-protoZoal drug mentally different than bacteria and viruses. Unlike bacteria Would target multiple protoZoan parasites, be active by vari and viruses, protoZoa parasites are animals and share similar ous routes of administration, reduce morbidity and mortality metabolism, respiration, and nutritional needs With their ani 45 caused by such infections, not interfere With co-administered mal hosts. The similar metabolism of protoZoa to mammalian vaccines as they become available, and reduce the need for metabolism renders most antibiotics and antiviral agents, hospital-based treatment. selectively active against bacteria and viruses, respectively, The important protoZoal sources of infection addressed by ineffective for protoZoal infection. Activity of compounds the methods of treatment and compositions of the present With antibacterial or antiviral activity against protoZoal para 50 invention are a subset of protoZoal organisms Within the bio sites Would be atypical and unexpected. The lack of differ logic kingdom Protista. The protoZoa relevant to the present ences betWeen protoZoal metabolism and host cellular invention are summarized in Table 1. TABLE 1

Selected ?eld of parasites relevant to treatment methods and compositions using DIM-related indoles.

Phylum Class Order Genus Species

Chromista Microsporidia Parabasalea Trichomonadida Enlerocylozoon bieneusi Parabasalia Bigyra Trypanosomatida Leishmania Blaslocysl‘is hominis Euglenozoa Axostylata Adeleida Trypanosoma Trichomonas vaginalis Subphylum Kinetoplasta Eimeriida Cysloisospora Leishmania species Apicomplexa (Sporozoa) Trypanosomatidea Haemosporida Plasmodium donovani, infanlum (chagasi), Coccidea Conoidasida Eucoccidiorida lropica, braziliensis, guyanensis Trypanosoma species Brucei, gambiense, rhodesiense US 8,586,621 B2

TABLE l-continued

Selected ?eld of parasites relevant to treatment methods and compositions using DIM-related indoles.

Phylum Class Order Genus Species Trypanosoma cruzi Trypanosoma rangeZi Cryplosporidium baiZeyi Cryplosporidium meleagridis Cryplosporidium parvum Cryplosporidium muris CycZospora cayelanensis Isospora belli Cysloisospora Toxoplasma gondii Plasmodiumfalczparum Plasmodium malariae Plasmodium ovale Plasmodium vivax Babesia gibsoni Babesia microli Neospora Sarcosysris homins Suihominis, Zindemanni

2.2 Scope of Protozoal Parasitic Infections ness. Treatment of CM is limited to a feW conventional anti

25 malarial drugs (quinine or artemisinins) and supportive care Parasitic protozoa are responsible for a variety of human including parenteral ?uids, blood exchange transfusion, diseases transmitted by insect vectors, i.e., carriers, including osmotic diuretics and correction of hypoglycemia, acidosis malaria, leishmaniasis, and trypanosomiasis. Other protozoal and hypovolemia. The management of CM includes prompt parasites can be transmitted directly from other mammalian administration of appropriate parenteral anti-malarial agents reservoirs or from person to person. Lacking vaccines, vector 30 and early recognition and treatment of the complications. In control and selective chemotherapy have been the only Ways children, the complications include severe anemia, seizures to reduce transmission and treat infected individuals, respec and raised intracranial pressure. In adults, renal failure and tively. Because the immune system plays a crucial role in pulmonary edema are more common causes of death. controlling protozoal infection, opportunistic infection With A number of drugs ranging from those of natural origin to protozoal organisms is an increasing problem in infants, can 35 synthetic ones have been developed for the treatment of cer patients, transplant recipients, and those co-infected With malaria. Quinine and artemisinin are the commonly knoWn human immunode?ciency virus (HIV). Pregnancy also sup drugs of natural origin, Which are used for the treatment of presses certain immune functions. NeW anti-protozal treat malaria. A number of synthetic anti-malarial drugs such as ments are needed Which are safer for mother and fetus during chloroquine, me?oquine, primaquine, halofantrin, amodi pregnancy, particularly for malaria, toxoplasmosis, and tri 40 aquine, proguanil, atovaquone, maloprim are knoWn in the chomonas infections. Vaccines are needed Which overcome literature. Quinidine Gluconate, Quinine Sulfate, typically in diminished immune responses and induce an adequate long combination With Doxycycline hyclate, Clindamycin, or term immune response. Vaccines can be used in conjunction Pyrimethamine-sufadoxine are also used for malaria. In chlo With compatible chemotherapy to improve therapy of pre roqine resistant strains, preferred oral therapy includes existing chronic infection in endemic areas. 45 Me?oquine Hydrochloride and Atovaquone-proguanil 2.2.1 Malaria is an Uncontrolled Protozoal Disease hydrochloride combinations. In treatment of infections With Malaria arises from infection With an Apicomplexan pro R vivax, R malariae, R ovale, and chloroquine sensitive R tozoan parasite knoWn as Plasmodium. Only four species of falciparum, chloroquine phosphate and primaquine phos the genus Plasmodium cause human malaria. R vivax is the phate are used. most common and fatal. R ovale and R malariae are less 50 In recent years, drug resistant malaria has become one of common and have intermediate severity. P falciparum is the the most serious problems in malaria control. Drug resistance most virulent, responsible for high infant mortality, and asso necessitates the use of drugs Which are more expensive and ciated With current drug resistance. The disease is transmitted may have dangerous side effects. The emergence of resis to human beings through the bite of infected female Anoph tance can be prevented by the use of combinations of drugs eles mosquitoes and by transfusion of infected blood. 55 With different mechanisms of action. The use of drug combi Due to the emergence and spread of drug-resistant malaria nations for all antimalarial treatment not only delays the onset parasites, pesticide-resistant malaria-transmitting mosqui of drug resistance, but also accelerates recovery and increases toes, and population groWth in endemic areas, malaria noW cure rates. A number of antimalarial combinations are already causes approximately 500 million clinical cases per year. It is knoWn in the ?eld of malarial chemotherapy. The speci?c prevalent in children and pregnant Women, causing about one 60 combinations in use, dosages, and relative merits of various million annual deaths in children under the age of ?ve. Chil combinations have been summarized (Kremsner et al., 2004, dren groWing up in rural and endemic areas are subject to Lancet 364:285-94). more frequent malaria related illness and deaths than more With the emergence of R falciparum strains resistant to resistant adults. chloroquine and quinine, further alternative antimalarial che The most severe form of Plasmodium falciparum infection 65 motherapy is required. Due to frequent re-infection folloWing is cerebral malaria (CM). Cerebral malaria implies the pres complete or partial treatment, vaccine therapy promoting ence of neurological features, especially impaired conscious long term immunity to re-infection is needed. NeW chemo US 8,586,621 B2 5 6 therapy Will preferably clear the current infection and not protoZoan parasite Leishmania and transmitted by female interfere With co-admini stered vaccines as they become avail sand?ies. The disease is currently estimated to affect some 12 able. Preferred combinations of anti-malarials utilize drugs million people in 88 countries. WorldWide, leishmania/HIV that overcome chloroquine resistance, have a good safety co-infection is noW considered an emerging disease Where pro?le, and are Well tolerated. Artemisinin, obtained from the about 50% of adult visceral leishmaniasis cases are related to plantArZemisia anua, and its derivatives are rapidly effective co-existing HIV infection. in severe malaria. Artemisinin compounds have been evalu The current treatment for leishmaniasis involves adminis ated in several centers and are found to be effective, and safe tration of pentavalent antimony complexed to a carbohydrate (Miskra et al., 1995, Trans R Soc Trop Med Hyg 89:299-301). in the form of sodium stibogluconate (Pentosam or Sb(V)) or In addition, the patent literature describes the combination meglumine antimony (Glucantine), Which are the only estab of atovaquone and proguanil as a method for the treatment of lished anti-leishmanial chemotherapeutic agents With a malaria. See U.S. Pat. No. 5,998,449. The combination of clearly favorable therapeutic index. The exact chemical struc fenoZan With another anti-malarial agent selected from arte ture and mode of action of pentavalent antimonials is still misinin, sodium artesunate, chloroquine, or me?oquine is uncertain. Amphotericin B and Pentamidine are the second described for the prophylactic and curative treatment of line of anti-leishmanial agents, but are reserved for non malaria. See U.S. Pat. No. 5,834,505. Synergistic combina responding infections due to potential toxicity. Since resis tion kits using atemisinin derivatives, sulfadoxin and tance to the antimony-based anti-Leishmanial drugs is pyrimethamine for severe, multi-drug resistant malaria are emerging and treatment failures are common, neW combina described by Tipathi et al. in U.S. Patent Application Publi tion therapies are needed. Miltefosine is a recently introduced cation No. 2006/0141024 A1. 20 oral drug effective for visceral and cutaneous disease. The 2.2.2 Trypanosomiasis Lacks Effective Chemotherapy for importance of this neW oral agent extends to the treatment of Early and Late Disease dogs Which serve as an important reservoir of the disease. The African trypanosomiasis (sleeping sickness) is caused by a identi?cation of additional, neW and effective anti-leishma subspecies of the parasitic haemo?agellate, Trypanosoma nial agents for oral administration Would alloW further treat brucei. The infection begins With the bite of an infected tsetse 25 ment options, help prevent emerging resistance to Miltefos ?y (Glossina spp.). TWo forms of the disease are knoWn, one ine and antimony-based drugs, and increase the chance for caused by Trypanosoma brucei rhodesiense, endemic in regional control of leishmaniasis. DIM has been shoWn to be Eastern and Southern Africa, and the other caused by T b. a potent inhibitor of Leishmania donovani topoisomerase I gambiense, originally detected in West Africa, but also Wide (LdTOPILS) With an IC5O of 1.2 uM. See Roy A., et al., spread in Central Africa. African Trypanosomiasis results in 30 Biochemical Journal, 8 Oct. 2007, Immediate Publication febrile, life-threatening illness in humans and also threatens Manuscript B] 20071286 (not the ?nal version). livestock. T brucei parasites rapidly invade the Central Ner 2.2.4 Trichomonal Disease vous System (CNS) causing death Within Weeks if untreated. Trichomonal infection, typically vulvo-vaginitis in Women T b. gambiense proliferates relatively sloWly and can take and urethritis in men, is sexually acquired and one of the most several years before infecting the CNS system. There are four 35 common protoZoal parasite infections in humans. In the important drugs approved to treat these infections. TWo of United States, it is estimated that more than 2 million Women these, pentamidine and suramin, are used before the CNS are infected each year. Trichomonas vaginitis causes vulvar involvement. The arsenic-based drug, melarsoprol is used in itching and an odorous vaginal discharge. It is caused by the case of infections established in the CNS. The fourth drug, Trichomonas vaginalis, a single-celled protoZoan parasite not e?ornithine, is used against late stage infection caused by T b. 40 normally found in the ?ora of the genitourinary tract. Typi gambiense. This drug is ineffective against T b. rhodesiense. cally Trichomonal infection is treated With oral metronida Nifurtimox is another drug licensed for both American try Zole Which is FDA approved in various dosage regimens. panosomiasis and melarsoprol-refractory late stage disease. Though ef?cacious, can exhibit serious dose American trypanosomiasis or Chaga’s disease is caused by related side effects, particularly on the blood and on the Trypanosoma cruzi and effects millions of people in South 45 central nervous system. Experiments shoW it to be mutagenic and Central America, and Mexico. Untreated Chaga’ s disease and carcinogenic. Recently, treatment failure and emerging causes decreased life expectancy due to parasitic cardiomy resistance to metronidiZole have been documented, indicat opathy and heart failure, megaesophagus, and megacolon. ing a need for more consistently effective therapies Which Will Blood-sucking triatomid bugs transmit the infection to young include combinations of drugs active against strains of T children and transplacental infection can occur With para 50 vaginalis that may be resistant to metronidaZole. Preferred sitemia during pregnancy. Nifurtimox and benZnidaZole are treatments Will include agents safe for pregnant Women and tWo drugs used for treatment of the acute disease, but are not alloW loWer doses of co-administered metronidaZole. knoWn to be therapeutic for the chronic infection in older 2.2.5 ProtoZoal Disease in Immunocompromised Hosts children and adults. In the absence of an effective vaccine, The risk of parasitic diseases is also present outside devel better agents are needed that can be taken prophylactically by 55 oping countries and often takes the form of chronic diarrheal at risk children. Following infection, additional agents are disease in subjects With underlying immune de?ciency. These needed to be used in conjunction With nifurtimox and ben infections can be caused by Isospora belli, and Cyclospora ZnidaZole to increase e?icacy, permit loWer doses of the cur cayelanensis, both coccidian protoZoa, Where infection rent agents With reduced toxicity, and shorten the currently results in self-limited diarrhea in normal hosts and prolonged required duration of treatment. 60 diarrhea in individuals With AIDS. Both infections respond to 2.2.3 Leishmaniasis Lacks Practical and Safe Chemo treatment With timethroprim-sufamethoxaZole. Cryptospo therapy ridia are additional coccidian parasites that cause diarrhea in Human leishmaniasis comprises a heterogeneous spec animal species and humans. Cryplosporidium parvum and C. trum of diseases. Three major forms are generally distin Hominis account for most coccidial infections in humans. guished: cutaneous leishmaniasis, mucocutaneous leishma 65 These organisms form oocytes, Which When digested release niasis and visceral leishmaniasis, of Which the latter is sporoZoites that invade host epithelial cells, penetrating the potentially lethal. They are caused by various species of the cell membrane but not the enterocyte cytoplasm. NitaZox US 8,586,621 B2 7 8 anide is the only drug approved for the treatment of (Hong et al., 2002, Biochem Pharmacol. 63: 1085-97). In cryptosporidiosis in the United States. The identi?cation of animals, orally administered DIM inhibits the groWth of cer additional effective anti-crytosporidial agents for oral use tain chemically induced forms of breast cancer (Chen et al., Would alloW additional treatment options for individuals With 1998, Carcinogenesis 19:1631-9). Recently, DIM has been HIV infection Who respond unpredictably to NitaZoxanide. shoWn to speci?cally induce apoptosis in Human Papilloma Toxoplasmosis, is a Zoonotic infection by the obligate Virus (HPV) oncogene altered cervical cancer cell lines intracellular protozoan, Toxoplasma gondii. Toxoplasmosis (Chen et al., 2001, J Nutr. 131:3294-3302). In further cell is found throughout the World, including the United States. culture experiments, DIM has been shoWn to reduce activa Cats and other feline species are the natural hosts for Toxo tion of the NFKB signaling pathWay in breast cancer cells plasma gondii, hoWever tissue cysts (bradyZoites) have been (Rahman et al., 2005, Cancer Res. 65:364-71). Other non recovered from all mammalian species examined. Pregnant DIM I3C condensation products Were not tested. In vivo Women and those With Weak immune systems are particularly studies in mice suggest that expected effective plasma levels susceptible to the health risks resulting from Toxoplasma of DIM are not easily achieved in humans (Anderton et al., infection. Severe toxoplasmosis, particularly trans-placental 2004, Drug Metab Dispos. 32:632-8). exposure, can result in damage to the brain, eyes, and other In relation to its pro-apoptotic activity in tumor cells, DIM developing organs in utero. Currently available treatments for has also been shoWn to be estrogenic in breast cancer cells toxoplasmosis, Which are the drugs trisulfa-pyrimdine, sulfa (Riby et al., 2000, Biochem. Pharmacol. 60:167-177) and in diaZine and pyrimethamine, are not effective, and can be toxic rainboW trout, a model of carcinogenesis relevant to cancer in to the ho st. Therefore, there is a need for therapeutic agents to humans (Shilling et al., 2001, Toxicology and Applied Phar treat toxoplasmosis that are more effective and less toxic than 20 macology 170: 1 91-200). Since estrogenic effects inhibit apo currently available treatment agents. No available agent is ptosis, DIM may actually enhance estrogen related groWth used to control Toxoplasmosis in cats. and survival of some cells. Based on the con?icting results of DIM’s activity in cell culture studies and estrogenic activity 2.3 ProtoZoal Cell Behavior Includes Apoptosis-Like in vivo, it is dif?cult to predict DIM’s effects in vivo on Responses and Suppression of Apoptosis in Infected 25 protoZoal disease processes. In addition, DIM has been Host Cells shoWn to activate the Mitogen Activated Protein Kinase (MAPK) cell signaling pathWay in cell culture (Leong et al., Apoptosis is the process of programmed cell death by 2004, Mol Endocrinol. 181291-302). Activated MAPK is Which damaged cells are eliminated upon generation of unop associated With cancer promotion, cancer cell survival, and posed death signals Within the damaged cell. While apoptosis 30 inhibition of apoptosis. These properties of DIM suggest that is primarily vieWed as a biologic response of multicellular DIM Would not be useful as a promoter of apoptosis in pro organisms providing a means of eliminating infected or trans toZoal infection. formed cells in the setting of viral and cancer-related disease, One approach, that has not been developed for protoZoal protoZoal organisms have also been noted to exhibit pro parasitic disease, Would be to selectively induce apoptosis in grammed cell death behavior (Lee et al., 2002, Cell Death 35 protoZoal infected cells and tissues in order to cause the Differ. 9:53-64). When infecting host cells in mammals, pro programmed death of parasites and of parasite infected cells. toZoal parasites have also been noted to suppress host cell This Would result in parasite clearance and increased apop apoptosis. For example, activation of the Nuclear Factor tosis may support the development of protective host immu Kappa B (NFKB) survival signaling pathWay has been n1ty. described folloWing infection by Trypanosoma cruzi (Pe 40 tersen et al., 2006, Infect Immun. 74:1580-7). 3. SUMMARY OF THE INVENTION

2.4 Natural Indole Compounds can In?uence The present invention relates to Diindolylmethane (DIM) Apoptosis and DIM-related indoles that are useful for the treatment and 45 prevention or reducing the risk of protoZoal diseases in mam Cruciferous vegetables contain a family of plant protective mals and birds. The present invention also relates to compo compounds called glucosinolates Which give rise to active sitions comprising DIM-related indoles, optionally, in com compounds With indole rings exempli?ed by indole-3 bination With one or more additional antiprotoZoal agents. In carbinol (I3C). Oral ingestion of BC results in the gastric certain embodiments, the compounds and methods of the conversion of BC into at least tWenty acid condensation 50 present invention are used for treatment or prevention or products, many of Which are bioavailable, the most prevalent reducing the risk of infections by the primary protoZoal para of Which include CTR (cyclic trimer; 5,6,11,12,17,18 sites affecting humans, including, but not limited to, hexahydrocyclonona[1,2-b:4,5-b':7,8-b"]triindole), HI-IM hemo?agellates of the class Trypansomatidae, causing leish (1 - (3 -hydroxymethyl)-indolyl -3 -indolylmethane), DIM (di maniasis and trypanosomiasis, ?agellates of the class Axo indolylmethane), ICZ (indolocarbaZole) and LTr-1 (linear 55 stylata causing Trichomonal infection, and parasites of the trimer; [2 - (indol-3 -ylmethyl)-indol-3 -yl] indol-3 -ylmethane) phylum, Apicomplexa, causing malaria, toxoplasmosis, (Stresser et al., 1995, Drug Metabolism and Disposition cryptosporidiosis, and bebesiosis. Also covered by the meth 231965-975). The fact that there are many non-DIM acid ods are treatment of infections by fungi of the genus Candida, condensation products of BC, produced in vivo at equal or particularly C. albicans, Which behave like intracellular pro greater levels as DIM, Which can be responsible for I3C’s 60 toZoa by invading epithelial cells. In certain embodiments, activity, requires that biologic activities of individual conden the compounds and methods of the present invention are used sation products like DIM be demonstrated directly. for treatment or prevention or reducing the risk of infections As one of many products derived from BC, DIM is also affecting non-human mammals, including, but not limited to, present in cruciferous plants folloWing release of BC. Once protoZoal parasitic diseases functionally related to malaria formed, DIM is stable in acid. In cell culture, isolated DIM 65 caused by parasites of the species Neospora. Neospora infec has been shoWn to have apoptosis promoting effects in both tions are knoWn to occur in dogs, cattle, sheep, goats and estrogen-dependent and independent breast cancer cells horses. In certain embodiments, the compounds and methods US 8,586,621 B2 10 of the present invention are used for treatment or prevention acid, campothecins, especially topotecan, irinotecan, SN38 or reducing the risk of infections With species of Coccidea, (the active metabolite of irinotecan), borteZimib, etoposide, also of the phylum Apicomplexa, Which infect humans, salvicine, and doxorubicin. AntiprotoZoal natural products domestic animals and livestock, including poultry, lambs, useful With the methods of the present invention include teas calves, piglets, and rabbits. As used herein, a subject includes, and extracts made from Arlemisia annua, teas and extracts but is not limited to, humans, domestic animals such as dogs, made from Curcuma domeslica, extracts from garlic Which and livestock such as poultry, sheep, lambs, piglets, rabbits, include allicin and other thiosul?nates, root extracts of cattle, calves, goats and horses. In certain embodiments, the Uvaria chamae (Annonaceae) and Hippocralea A?’icana compounds and methods of the invention are used for the (Hippocrateaceae), and root extracts of Homalium leleslui. treatment or prevention or reducing the risk of infection Preferred agents for combined use With DIM-related indoles caused by Cyclospora, Isospora, or Blaslocyslis. include artemisinin extracts and related drugs, and In certain embodiments, the methods of the invention curcumin-related drugs, and other antiprotoZoal agents With employ structurally-related, synthetically-derived, substi short metabolic half lives. Speci?c, preferred antimalarials tuted diindolylmethane compounds referred to as DIM-re With rapid metabolism include artesunate, dihydroartemisi lated indoles to treat protoZoal parasite infections. In a par nin, quinine, and clindamycin. ticular embodiments, the one or more DIM-related indoles of In a particular embodiment, the DIM-related indole and an the invention are selected from the group consisting of 3,3‘ antiprotoZoal agent are administered simultaneously. In diindolylmethane (DIM), hydroxylated DIMs, methoxylated another embodiment, the DIM-related indole and antiproto DIMs, 2-(Indol-3-ylmethyl)-3,3'-diindolylmethane (LTR), Zoal agent are administered Within a short time of one another, hydroxylated LTRs, methoxylated LTRs, 5,5'-dimethylDIM 20 for example, 30 seconds, 1 minute, 5 minutes, 15 minutes, 30 (5-Me-DIM), 2,2'-dimethylDIM (2-Me-DIM), 5,5'-dichlo minutes, 1 hour, 4 hours, 8 hours, 12 hours or 24 hours of one roDIM (5-Cl-DIM), imidaZolyl-3,3'-diindolylmethane, another. nitro-substituted imidaZolyl-3,3'-diindolylmethanes, 2,10 In an additional embodiment, the combination of a DIM dicarbethoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carba related indole and a antiprotoZoal agent is administered in Zole, 6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]car 25 conjunction With a differentiation promoting agent Which baZole and 2,l0-dicarbethoxy-6-ethoxycarbonyloxy-5,7 helps protoZoa infected cells develop into more completely dihydro-indolo-[2,3-b]carbaZole, and 2,6-dicarbethoxy-3,3' differentiated and therapeutically sensitive cells. Differentia dimethyl-l3,14-diindolylmethane, and indole-3 carbinol tion promoting agents include Vitamin-D, Vitamin-D deriva (BC). tives,V1tamin-A (retinoids), 9-cis-Retinoic acid, l3-cis-Ret In certain embodiments the DIM-related indole or indoles 30 inoic acid, trans-Retinoic acid, all-trans-Retinol, retinyl are used in combination With anti-protoZoal therapeutics. acetate, Retinyl palmitate, and granulcyte/macrophage Anti-protozoal agents Which are combined With DIM-related colony stimulating factors including recombinant human indoles according to the present invention include established Filgrastim and Sargramostim. antiprotoZoal drugs, natural product derived antiprotoZoal In an additional embodiment, the combination of a DIM drugs, apoptosis promoting drugs often With a history of use 35 related indole and a antiprotoZoal agent is administered in as apoptosis promoting chemotherapeutics, and natural prod conjunction With anti-protoZoal vaccines Which contain ucts from plants With antiprotoZoal activity. Established anti attenuated protoZoal organisms, typically inactivated by irra protoZoal agents for combined use include atovaquone, amo diation and/or chemical processing. Alternatively, the DIM diaquine, amphotericin, butoconaZole, clindamycin, related indole, antiprotoZoal agent, and anti-protoZoal vac e?ornithine, fumagillin, iodoquinol (diiodohydroxyquin), 40 cine, are administered With an additional immune (iodochlorhydroxyquin), EtanidaZole, BenZnida potentiating agent (Which is not a DIM-related indole). Zole, ?uoroquinolones, enoxacin, cipro?oxacin, doxycy Immune potentiating agents useful in the methods of the cline, melarsoprol, metronidaZole, miltefosine, nifurtimox, present invention include aloe vera extracts, puri?ed aloe nitaZoxanide, , pentamindine, sodium stiboglu mannans and acemanans, mushroom extracts, beta-glucans, conate, suramin, tinidoZole, pyrimethamine, proguanil (chlo 45 and extracts of the root of North American ginseng (Panax roguanide), spiramycin, and sulfadoxine. Natural product quinquefolium) containing poly-furanosyl-pyranosyl-sac derived antiprotoZoal drugs useful for combined use include charides (CV Technologies Inc., Edmonton). Beta-glucans sesquiterpene lactones related to artemisinin from Arlemisia include those derived from Saccharomyces cerevisiae (Im annua, particularly artemisinin, dihydroartemisinin, arte munDyne, Inc., Florence, Ky.). Other useful fungal extracts mether, artesunate, and further derivatives of artemisinin, 50 containing branched glucans are derived from mushrooms, quinolines like quinine derived from the bark of the South such as the maitake mushroom (Grifolafrondosa). American chinchona tree, including quinine and quinine The invention further provides compositions, for example, related quinolines, halofantrine, me?oquine, lumefantrine, a composition comprising a therapeutically effective amount amodiaquine, pyronaridine, piperaquine, chloroquine, of the combination of DIM, or a DIM-related indole, and an hydryoxychloroquine, napthoquine, primaquine, and tafeno 55 anti-protoZoal compound or combination of anti-protoZoal quine, curcuminoids derived from curcumin, an extract from compounds. In particular embodiments, the compositions are Curcuma domeslica, including 6-gingerol and 6-paradol, formulated for oral, sublingual, rectal, vaginal, parenteral, coronaridine, l8-methoxycoronaridine, selected ?avonoids, and topical administration. In a further particular embodi including luteolin, extracts the fruit pericarp of Sapindus ment, the different formulations are combined to form a kit mukorossi, and extracts of Yucca schidigera. Apoptosis pro 60 combining rectal suppositories With an oral suspension for moting antiprotoZoal agents for combined use include arte pediatric use, or rectal or vaginal suppositories With capsules misinin derivatives, atovaquone, chloroquine, iodoquinol (di or tablets for adult use. iodohydroxyquin), clioquinol (iodochlorhydroxyquin), The present invention also provides compositions for treat sodium stibogluconate, and curcumin. Some apoptosis pro ing protoZoal infections comprising DIM, a DIM-related moting chemotherapeutics used are also useful in combina 65 indole, or DIM in combination With selected knoWn antipro tion With DIM-related indoles and include PyrroloquinaZo toZoal compounds, in an amount effective to reduce blood, linediamine, Novobiocin, cyclosporine, dihydrobetulinic tissue or intestinal parasite counts, formulated in the form of US 8,586,621 B2 11 12 a dietary supplement, for example, a nutraceutical tablet, parasites utiliZe a variety of strategies to avoid interaction capsule, drink mix, or forti?ed food; a tea mix, or chewing With the ho st immune system including differentiation to less gum. activating forms, inhibition of pro-apoptotic stress proteins, In the methods and compositions of the invention, DIM or and inhibition of immune activating antigen display on the a DIM-related indole is preferably processed to increase bio surface of host cell membranes. DIM-related indoles trigger availablity and/ or microencapsulated With phosphatidylcho pro-apoptotic signals through endoplasmic reticulum stress line (PC), complexed With PC, or made into rapidly dissolv Which has been shoWn to induce apoptosis in cancerous cells ing microparticles and nanoparticles. Preferably, the (Sun et al., 2004, Cell Stress Chaperones. 9(1):76-87). formulations Will include specialiZed vehicles for rectal and Without being bound by any theory, the present invention vaginal administration and be safe for use during pregnancy. employs DIM-related indoles alone and together With addi tional antiprotoZoal agents to inhibit the protoZoal parasite 4. DETAILED DESCRIPTION OF THE associated activation of NFKB and selectively induce apop INVENTION tosis in parasite infected cells, thereby reducing production of protoZoal progeny, reducing parasite load, and resolving or The present invention relates to Diindolylmethane and shortening the period of infection. Prophylactic uses of DIM DIM-related indoles that are useful for the treatment and related indoles alone or With antiprotoZoal agents can prevent prevention or reducing the risk of protoZoal diseases in mam primary infection or re-infection With protoZoal parasites. mals and birds. In particular, the invention relates to the Selective inhibition of overactive survival and groWth signals treatment and prevention of the protoZoal diseases in mam 20 in protoZoal parasite infected cells in the present invention mals and birds in Table 1. In certain embodiments, a DIM can provide effective therapy, causing protoZoa altered cells related indole is used in combination With one or more of the to be eliminated by triggering programmed cell death (apop following: an anti-protoZoal agent, a differentiation agent, an tosis). Importantly, the promotion of apoptosis in protoZoa immune potentiating agent and an anti-protoZoal vaccine. infected cells can stimulate a more effective immune The present invention is directed to compositions comprising 25 response, enhancing natural immunity and improving the a DIM-related indole, either alone or in combination With one short and long term bene?t from co-administered anti-proto or more additional antiprotoZoal agents. The compounds and Zoal vaccines (James, 2005, J Infect Dis. 191(10):1573-5). methods of the present invention may be used for treatment In certain embodiments, these methods of the present and control of infections by the primary protoZoal parasites invention employ structurally-related, synthetically-derived, affecting man, including, but not limited to, hemo?agellates 30 substituted diindolylmethane compounds administered of the family Trypansomatidae, causing Leishmaniasis, Try orally, parenterally, vaginally, or per rectum. In a particular panosomiasis, and Bebesiosis, ?agellates of the class Axo embodiment, a combination of DIM, or a DIM-related indole, stylata causing Trichomonal infection, and parasites of the and one or more knoWn anti-protoZoal agents are provided. SporoZoa phylum, Apicomplexa, causing malaria, toxoplas The methods and compositions provide improved treatment mosis, and cryptosporidiosis. Also covered by the methods for protoZoal parasite infections. are treatment of infections by fungi of the genus Candida, The invention is based in part on expected additive and particularly C. albicans, Which behave like intracellular pro synergistic activity in using particular combinations of DIM toZoa by invading epithelial cells. In non-human mammals, related indoles and antiprotoZoal agents to selectively pro protoZoal parasitic disease functionally related to malaria 40 mote apoptosis in protoZoal infected cells and apoptosis-like includes disease caused by parasites of the species Neospora. cell death in extracellularparasites. Combined use With DIM Neospora infections are knoWn to occur in dogs, cattle, sheep, related indole is believed to permit loWer dose use of antipro goats and horses. Also treatable according to the methods and toZoal agent(s), reducing dose-related side effects of these compositions of the present invention is infection With spe drugs. In certain embodiments, the compositions of the inven cies of Coccidea, also of the phylum Apicomplexa, Which 45 tion can be used With differentiation promoting agents such as infect humans, domestic animals and livestock, including Vitamin-D derivatives (calcitriol[1-alpha-25-dihydroxyc poultry, lambs, calves, piglets, and rabbits. holecalciferol]), retinoid derivatives (Vitamin-A, isotretin Without being bound by any theory, it is believed that the oin, retinoids), macrophage colony stimulating factors compounds and methods of the present invention promote (Filgrastim and Sargramo stim), and other immune potentiat apoptosis, for example, by to inhibiting cell survival signaling 50 ing agents. The combination of a DIM-related indole and in host cells Where such signaling is a response protoZoal antiprotoZoal agents is believed to induce promotion of apo parasite infection, Which supports the induction of host ptosis resulting in the selective elimination of protoZoa immunity, especially When used in conjunction With anti infected cells, and causes resolution of protoZoal parasite protoZoal vaccines. Promotion of more e?icient apoptosis 55 related lesions of infected tissues, particularly the intestines, and interaction of protoZoal antigens With host immune cells liver, skin, heart, spleen, and blood. is believed to result from the combined use of DIM-related indole With a variety of anti-protoZoal agents, especially anti 4.1 Diindolylmethane-Related Indoles protoZoal vaccines.

ProtoZoal infection of cells initiates cell-groWth and cell 60 The DIM-related indoles or DIM compounds useful in the survival mechanisms uniquely attributed to the interaction of methods and compositions of the invention include DIM protoZoal parasite With host cell apoptotic mechanisms (3,3'-diindolylmethane) and the related linear DIM trimer (Heussler et al., 2001, Int J Parasitol. 31:1166-76). Upon (2-(indol-3-ylmethyl)-3,3'-diindolylmethane [also Written: 2 entry into cells, protoZoal parasites provide an activation sig (Indol-3 -ylmethyl)-indol -3 -yl] indol -3 -ylmethane] (LTR), nal for cell survival including activation of NFKB signalling 65 and Indole-3-Carbinol (I3C). As used herein, “DIM-related Which inhibits cellular apoptosis (Shapira, 2004, J Parasitol. compound”, “DIM-related indole”, and “DIM derivative” are 34(3):393-400). In protoZoal parasite infections, protoZoal used interchangeably, and refer to both natural metabolites US 8,586,621 B2 13 14 and analogs of DIM, and also to “structurally-related, syn In particular embodiments, the indolyl moieties are sym thetically-derived, substituted diindolylmethane com metrically substituted, Wherein each moiety is similarly pounds” and “synthetic derivatives of DIM”, such as those mono-, di-, tri-, para-, etc. substituted. In other particular disclosed herein and known in the art. As used herein, “cru embodiments, R42, R51, R35, R37, R38, R90, R41, R50, R31, ciferous-related indoles” encompasses the terms “DIM-re R33 , R34 and R91 are hydrogen, and R3 6 and R32 are a halogen lated compound”, “DIM-related indole”, and “DIM deriva selected from the group consisting of chlorine, iodine, bro tive”. One of ordinary skill in the art Will recognize that in any mine and ?uorine. Representative compounds include, but of the pharmaceutical compositions or methods of the inven are not limited to, 3,3'-diindolylmethane, 5,5'-dichloro-diin tion Where DIM is used, a DIM-related compound, including dolylmethane; 5,5'-dibromo-diindolylmethane; and 5,5'-dif a structurally-related, synthetically-derived, substituted diin luoro-diindolylmethane. Additional preferred such DIM dolylmethane compound or synthetic derivative of DIM, can derivatives include compounds Wherein R42, R51, R35, R37, be used. R38, R90, R41, R50, R31, R33, R34 and R91 are hydrogen, and The chemical structure of a DIM is as folloWs (Where each R36 and R32 are an alkyl or alkoxyl having from one to ten of the R groups is H): carbons, and most preferably one to ?ve carbons. Represen tative compounds include, but are not limited to, 5,5'-dim ethyl-diindolylmethane, 5,5'-diethyl-diindolylmethane, 5,5' (I) dipropyl-diindolylmethane, 5,5'-dibutyl-diindolylmethane, 20 5 ,5 '-dipentyl -diindolylmethane, 5 ,5 ' -dimethoXy-diindolyl - methane, 5,5'-diethoxy-diindolylmethane, 5,5'-dipropyloxy diindolylmethane, 5,5'-dibutyloxy-diindolylmethane, and 5,5'-diamyloxy-diindolylmethane. Additional preferred DIM derivatives include compounds 25 wherein R51’ R35’ R36, R37’ R38, R90’ R50’ R31’ R32’ R33’ R34 and R91 are hydrogen, and R42 and R"1 are an alkyl or alkoxyl having from one to ten carbons, and most preferably one to ?ve carbons. Representative compounds include, but are not In particular embodiments, the DIM-related indole is a limited to, N,N'-dimethyl-diindolylmethane, N,N'-diethyl compound of formula I, Wherein R42, R5 1, R35, R3 6, R37, R38, 30 diindolylmethane, N,N'-dipropyl-diindolylmethane, N,N' R90, R41, R50, R31, R32, R33, R34 and R91 individually and dibutyl-diindolylmethane, and N,N'-dipentyl-diindolyl independently, are hydrogen or a substituent selected from methane. In yet another embodiment, R42, R35, R3 6, R37, R3 8, the group consisting of a halogen, a hydroxyl, a nitro, R90, R41, R31, R32, R33, R34 and R91 are hydrogen, and R51 iORIOO’ ‘CNS iNRIOO’ R101’ iNRIOO’ R101’ RlO2+’ and R50 are alkyl of one to ten carbons, and most preferably iCORlOO, CF3, iS(O)nRlOO (n:0-2), iSOZNRIOO, R101, 35 one to ?ve carbons. Representative compounds include, but iCONRlOO’ R101’ iNRlOOCORlOl’ iNR100C(O)NR101’ are not limited to, 2,2'-dimethyl-diindolylmethane, 2,2'-di R102, iP(O)(ORlOO)n (n:1-2), optionally substituted alkyl, ethyl-diindolylmethane, 2,2'-dipropyl-diindolylmethane, halovinyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroaryl, or 2,2'-dibutyl-diindolylmethane, and 2,2'-dipentyl-diindolyl optionally substituted cycloalkyl or cycloakenyl, all of one to 40 methane. In another embodiment, R42, R51, R35, R37, R38, ten carbons and optionally containing 1-3 heteroatoms O or R90, R41, R50, R31, R33, R34 and R91 are hydrogen, and R36 N, Wherein R100, R101 and R102 are optionally substituted and R32 are nitro. alkyl, alkenyl, alkynl, aryl, heteroalkyl, heteroaryl of one to In an alternative embodiment, active DIM derivatives With ten carbons, and R90 and R91 may further be 0 to create a R32 and R36 substituents made up of ethoxycarbonyl groups, ketone. In particular embodiments, the compound includes at 45 and R50, R51 are either hydrogen or methyl, are utiliZed. least one such substituent, preferably at a position other than, In another embodiment, active substituted DIM derivatives or in addition to R42 and R41, the linear or branched alkyl or including methylated and chlorinated compounds, exempli alkoxy group is one to ?ve carbons, and/or the halogen is ?ed by those that include 5,5'-dimethylDIM (5-Me-DIM), selected from the group consisting of chlorine, iodine, bro 2,2'-dimethylDIM (2-Me-DIM), and 5,5'-dichloroDIM mine and ?uorine. 50 (5-Cl-DIM) are described in Us. Patent Application Publi In certain embodiments, an active hydroxylated or methy cation No. 20020115708 by Safe, published Aug. 22, 2002, oxylated metabolite of DIM, i.e., a compound of formula I, incorporated herein by reference in its entirety, are utiliZed in Wherein R3 2, R3 3 , R3 6, and R37 are sub stituents independently the present invention. In another embodiment, active DIM selected from the group consisting of hydrogen, hydroxyl, derivatives include imidaZolelyl-3,3'-diindolylmethane, and methoxy, and R31, R34, R35, R38, R41, R42, R50, and R51 55 including nitro substituted imidaZolelyl-3,3'-diindolyl are hydrogen, is utiliZed. In particular embodiments, the methanes, and additional DIM-related compounds described DIM-related indole is a mono- or di-hydroxylated DIM in Us. Patent Application Publication No. 2004/ 0043965 by derivatives at carbon positions 2, 4-7 and 2', and 4'-7', includ long, Ling, published Mar. 4, 2004, incorporated herein by ing each of [2, 4, 5, 6 or 7] -monohydroxy-DIM or [2', 4', 5', 6' 60 reference in its entirety, are utiliZed. In a further embodiment, or 7']-monohydroxy-DIM (e.g. 2-hydroXy-DIM, 4-hydroxy active DIM derivatives described in Us. Pat. No. 6,656,963, DIM, etc.); and each of [2, 4, 5, 6 or 7], [2, 4, 5, 6 or 7]-di U.S. Pat. No. 6,369,095 and Us. Patent Application Publi hydroXy-DIM, [2', 4', 5', 6' or 7'], [2', 4', 5', 6' or 7']-dihydroxy cation No. 20060229355 by Bjeldanes et al., published Oct. DIM, or [2, 4, 5, 6 or 7], [2', 4', 5', 6' or 7']-dihydroXy-DIM 12, 2006, incorporated herein by reference in its entirety, are (e. g. 2,4-dihydroXy-DIM, 2,5-dihydroXy-DIM etc, 2,2'-dihy 65 utiliZed. droXy-DIM, 2,4'-dihydroXy-DIM etc.); particularly bilater The chemical structure of LTR is as folloWs (Where each of ally symmetrical species, such as 2,2'-dihydroXy-DIM. the R groups is H): US 8,586,621 B2 15 16 R1 l and R12 are independently selected from the group (I1) consisting of hydrogen, C 1 -C24 alkyl, C2-C24 alkoxycarbo nyl, amino-substituted C l-C24 alkyl, (C l-C24 alkylamino) substituted C l-C24 alkyl, and di-(Cl-C24 alkyl)amino-substi tuted C 1 -C24 alkyl, With the provisos that: at least one of R1, R2, R3, R4, R5 , R6, R7, R8, R9, R10, R1 1 and R12 is other than hydrogen; and When R1, R2, R3, R4, R5, R6, R7, and R8 are selected from hydrogen, halo, alkyl and alkoxy, then R1 1 and R1 2 are other than hydro gen and alkyl. A preferred embodiment includes the use of 2, l0-dicarbe thoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carbaZole (SRI13668 (SRI Inc., Menlo Park, Calif.)). Additional pre ferred embodiments include the use of 6-ethoxycarbonyloxy 5 ,7-dihydro-indolo-[2,3 -b] carbaZole and 2,10-dicarbethoxy 6-ethoxycarbonyloXy-5,7-dihydro-indolo-[2,3-b]carbaZole (SRI Inc., Menlo Park, Calif). In another embodiment, a DIM related compound has for mula (IV): 20 In certain embodiments, an active hydroxylated or methy oxylated metabolite of LTR, i.e., a compound of formula II, (IV) Wherein R62, R63, R66, R67, R70, and R71 are substituents R2 R1 R5 R6 independently selected from the group consisting of hydro R13 R14 gen, hydroxyl, and methoxy, and R61, R64, R65, R68, R69, R72, 25 R3 R7 R81, R82, and R83 are hydrogen, is utiliZed. In certain embodiments, a DIM related compound has formula (III): R4 N X N R8 R/l l \Rl 2 30 (III) Wherein: R1, R2, R3, R4, R5, R6, R7, and R8 are substituents inde pendently selected from the group consisting of hydrogen, 35 Cl-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C5-C2O aryl, C6-C24 alkaryl, C6-C24 aralkyl, halo, hydroxyl, sulfhydryl, Cl-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2O aryloxy, acyl, acyloxy, C2-C24 alkoxycarbonyl, C6-C2O aryloxycarbonyl, halocarbonyl, C2-C24 alkylcarbon 40 ato, C6-C2O arylcarbonato, carboxy, carboxylato, carbamoyl, mono-(Cl-C24 alkyl)-substituted carbamoyl, di-(Cl-C24 Wherein: alkyl)-substituted carbamoyl, mono-substituted arylcarbam R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are substituents oyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato, independently selected from the group consisting of hydro isocyanato, isothiocyanato, aZido, formyl, thioformyl, amino, gen, Cl-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C5-C2O 45 mono- and di-(Cl-C24 alkyl)-substituted amino, mono- and aryl, C6-C24 alkaryl, C6-C24 aralkyl, halo, hydroxyl, sulfhy di-(CS-C2O aryl)-substituted amino, C2-C24 alkylamido, dryl, Cl-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2O arylamido, imino, alkylimino, arylimino, nitro, C5-C2O aryloxy, acyl, acyloxy, C2-C24 alkoxycarbonyl, nitroso, sulfo, sulfonato, Cl-C24 alkylsulfanyl, arylsulfanyl, C6-C2O aryloxycarbonyl, halocarbonyl, C2-C24 alkylcarbon Cl-C24 alkylsul?nyl, C5-C2O arylsul?nyl, Cl-C24 alkylsulfo ato, C6-C2O arylcarbonato, carboxy, carboxylato, carbamoyl, 50 nyl, C5-C2O arylsulfonyl, phosphono, phosphonato, phosphi mono-(Cl-C24 alkyl)-substituted carbamoyl, di-(Cl-C24 nato, phospho, phosphino, and combinations thereof, and alkyl)-substituted carbamoyl, mono-substituted arylcarbam further Wherein any tWo adjacent (ortho) substituents may be oyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato, linked to form a cyclic structure selected from ?ve-membered isocyanato, isothiocyanato, aZido, formyl, thioformyl, amino, rings, six-membered rings, and fused ?ve-membered and/or mono- and di-(Cl-C24 alkyl)-substituted amino, mono- and 55 six-membered rings, Wherein the cyclic structure is aromatic, di-(CS-C2O aryl)-substituted amino, C2-C24 alkylamido, alicyclic, heteroaromatic, or heteroalicyclic, and has Zero to 4 C6-C2O arylamido, imino, alkylimino, arylimino, nitro, non-hydrogen substituents and Zero to 3 heteroatoms, With nitroso, sulfo, sulfonato, Cl-C24 alkylsulfanyl, arylsulfanyl, the proviso that one but not both of R2 and R6 is amino, Cl-C24 alkylsul?nyl, C5-C2O arylsul?nyl, Cl-C24 alkylsulfo mono-substituted amino, or di-substituted amino; nyl, C5-C2O arylsulfonyl, phosphono, phosphonato, phosphi 60 R11 and R12 are independently selected from the group nato, phospho, phosphino, and combinations thereof, and consisting of hydrogen, Cl-C24 alkyl, C2-C24 alkoxycarbo further Wherein any tWo adjacent (ortho) substituents may be nyl, amino-substituted Cl-C24 alkyl, (Cl-C24 alkylamino) linked to form a cyclic structure selected from ?ve-membered substituted C l-C2 4 alkyl, and di-(Cl-C24 alkyl)amino-substi rings, siX-membered rings, and fused ?ve-membered and/or tuted C 1 -C24 alkyl; six-membered rings, Wherein the cyclic structure is aromatic, 65 R13 and R14 are de?ned as for R1, R2, R3, R4, R5, R6, R7, alicyclic, heteroaromatic, or heteroalicyclic, and has Zero to 4 and R8 With the proviso that at least one of R13 and R14 is other non-hydrogen substituents and Zero to 3 heteroatoms; and than hydrogen; and US 8,586,621 B2 17 18 X is O, S, arylene, heteroarylene, CRIS, R16 or NRl7 to be used With DIM-related indoles include Artemether wherein R15 and R16 are hydrogen, Cl-C6 alkyl, or together lumefantrine, artesunate and amodiaquine, dihydroartemisi form :CRls, R19 where R18 and R19 are hydrogen or Cl-C6 nin and piperaquine, artesunate and me?oquine, artesunate alkyl, and R17 is as de?ned for R11 and R12. and sulfadoxine-pyrimethamine, and dihydroartemisinin A preferred embodiment includes the use of 2,6-dicarbe napthoquine-trimetoprim. For crytosporidial infections thoxy-3,3'-dimethyl-13,14-diindolylmethane (SRI Inc., DIM-related indole, artemisinin and curcumin or genistein Menlo Park, Calif.). are a preferred combinations. For Trichomonal infections a In another embodiment, a DIM related compounds has DIM-related indole is used With artesunate, metronidaZole, formula (V): , or hexadecylphosphocholine (miltefosine). For Candidal infections a DIM-related indole is used With an aZole derivative, particularly an imidaZole compound like (V) clotrimaZole, miconaZole, or butoconaZole. For combined bacterial and Trichomonal vaginitis, a DIM-related indole is R2 R1 used in combination With clindamycin, or With both clinda R12 mycin and artesunate. Alternatively, multi-organism infec R3 X I! R8 tion can be treated With a DIM-related indole combined With metronidaZole, or With both metronidaZole and artesunate. 4.2.1 Anti-ProtoZoal Agents R7 Anti-protoZoal agents Which can be combined With DIM R4 IN R20 R21 20 related indoles according to the present invention include R1 1 established antiprotoZoal drugs, natural product derived anti R5 R6 protoZoal drugs, apoptosis promoting drugs often With a his tory of use as apoptosis-promoting chemotherapeutics, and Wherein: knoWn antiprotoZoal natural products. As used herein, an R1, R2, R3, R4, R5, R6, R7, R8, R11, R12, and X are de?ned 25 “anti-protoZoal agent” does not include a DIM-related indole as for compounds of formula (III); and (Which has anti-protoZoal activity). In other Words, in refer R20 and R21 are de?ned as for R1, R2, R3, R4, R5, R6, R7, ring to the combination of a DIM-related indole and an anti and R8. protoZoal agent, the “anti-protoZoal agent” is an agent other In yet another embodiment, the DIM-related indole is an than a DIM-related indole. indole-3-carbinol tetrameric derivative (Brandi et al., 2003, 30 AntiprotoZoal agents include, but are not limited, atova Cancer Res. 63:4028-4036). In a further embodiment the quone; diaminopyrimidines, especially amodiaquine, DIM-related indole is an indole-3-carbinol derivative amphotericin, butoconazole, astemizole clindamycin, e?o described as an anti-tumor agent (Weng J R, Tsai C H, Kulp mithine, fumagillin; the 8-hydroxyquinolines, iodoquinol S K, Wang D, Lin C H, Yang H C, MaY, Sargeant A, Chiu C (diiodohydroxyquin), clioquinol (iodochlorhydroxyquin), F, Tsai M H, Chen C S. A potent indole-3-carbinol derived 35 the 2-, EtanidaZole, BenZnidaZole ?uoroqui antitumor agent With pleiotropic effects on multiple signaling nolones, enoxacin, cipro?oxacin; doxycycline, melarsoprol, pathWays in prostate cancer cells. Cancer Res. 2007 Aug. 15; metronidaZole, tinidaZole, miltefosine, nifurtimox, nitaZox 67(16):7815-24). anide, paromomycin, pentamindine, sodium stibogluconate, Substituted DIM analogs are readily prepared by conden antimony gluconate (SAG), and related antimonials, suramin, sation of formaldehyde With commercially available substi 40 including the sodium salt, tinidaZole, pyrimethamine, tuted indoles. Precursor compounds can be synthesiZed by proguanil (chloroguanide), spiramycin, and sulfadoxine. dimethylformamide condensation of a suitable substituted Also useful are detergent and non-detergent spermacides that indole to form a substituted indole-3-aldehyde. Suitable sub have additional anti-protoZoal activity When used in topical stituted indoles include indoles having substituents at R42, formulations (Gupta G. Microbicidal spermicide or spermi R51, R35, R36, R37 and R38 positions. These include, but are 45 cidal microbicide? Eur J Contracept Reprod Health Care. not limited to 5-methoxy, 5-chloro, 5-bromo, 5-?uoro, 5'-me 2005 December; 10(4):212-8). thyl, 5-nitro, n-methyl and 2-methyl indoles. The substituted Natural product derived antiprotoZoal drugs include, but indole 3-aldehyde product is treated With a suitable alcohol are not limited to, sesquiterpene lactones related to artemisi such as methanol and solid sodium borohydride to reduce the nin from Arlemisia annua, particularly artemisinin, dihy aldehyde moiety to give substituted I3Cs. Substituted DIMs 50 droartemisinin, artemether, artesunate, and further deriva are prepared by condensing the substituted indole-3-carbinol tives of artemisinin described in the literature (Haynes, 2006, products. This may be achieved, for example, by treatment From artemisinin to neW artemisinin antimalarials: biosyn With a phosphate buffer having a pH of around 5.5-7.4. thesis, extraction, old and neW derivatives, stereochemistry and medicinal chemistry requirements. Curr Top Med. Chem. 4.2 Combination Therapy 55 6(5): 509-37); quinolines like quinine derived from the bark of the South American chinchona tree, including alkaloids In certain embodiments of the invention, a DIM-related structurally related to quinine, quinine and quine-related indole may be used in combination With one or more of the quinolines, halofantrine, me?oquine, lumefantrine, amodi folloWing: an anti-protoZoal agent, a differentiation agent, an aquine, pyronaridine, piperaquine, chloroquine, hydryoxy immune potentiating agent or an anti-protoZoal vaccine. It is 60 chloroquine, napthoquine, primaquine, tafenoquine, amodi understood that more than one agent from each class can be aquine and 4-aminoquinolines derived from the quinolines used, for example, multiple anti-protoZoal agents. (Neill et al., 2006, Curr Top Med Chem. 6:479-507); other Particularly preferred drug combinations to be used in quinones including those extracted from Salviaprionilis, par conjunction With additional DIM-related indoles according ticularly salvicine and its derivatives (Qing C. et al, In vitro to the present invention include the non-artemisinin combi 65 cytotoxicity of a salvicine, a novel diterpenoid quinone, nations quinine and sulfadoxine-pyrimethamine and quinine Zhongguo Yao Li Xue Bao. 1999 April; 20(4):297-302); cur and doxycycline. Artemisinin based combination treatments cuminoids derived from curcumin, extracted from Curcuma US 8,586,621 B2 19 20 domeslica, including 6-gingerol and 6-paradol (Surh et al., extracts, beta-glucans, and extracts of the root of North 1999, J Environ Pathol Toxicol Oncol. 18:131-9); selected American ginseng (Panax quinquefolium) containing poly ?avonoids and iso?avones, including, but not limited to, furanosyl-pyranosyl-saccharides (CV Technologies Inc., Genistein from soy, and derivatives from a number of plant Edmonton). Useful Panax quinquefolium extracts are sources including dehydrosilybin, silybinA and silybin B and described in Us. Pat. No. 6,083,932 by Pang et al. Which is isosilybin A and isosilybin B, and 8-(1; 1)-DMA-kaempfer herein incorporated by reference in its entirety. Beta-glucans ide (Tasdemir et al., 2006, Antimicrob Agents Chemother. include those derived from Saccharomyces cerevisiae (En 50:1352-64), luteolin, baicalein, dihydrobetulinic acid, quer Bio Technology Co., Ltd.). Other useful fungal extracts con cetin, eriodictyol acid, lursolic acid, oleanolic acid; and trit taining branched glucans are derived from mushrooms, such erpenes, particularly Ganoderic acid X, isolated from Gano as the maitake mushroom (Grifola frondosa). Oral use of derma amboinense and triterpene rich extracts of Sapindus beta-glucans in infections has been described (Jung et al., mukorossi knoWn to have anti-trichomonal activity. 2004, J Vet Med B Infect Dis Vet Public Health. 5 1 (2)172-6). Apoptosis promoting antiprotoZoal agents include, but are 4.2.4 Anti-ProtoZoal Vaccines not limited to, artemisinin derivatives, atovaquone, chloro Existing vaccines do not induce suf?cient protective quine, iodoquinol (diiodohydroxyquin), clioquinol (iodoch immunity to clear existing infection and prevent a repeat lorhydroxyquin), Jasmonic acid [3 -oxo-2-(2-pentenyl)cyclo infection. Therefore, in one aspect of the invention, a DIM pentaneacetic acid], methyl jasmonate[methyl 3-oxo-2-(2 related indole is administered With a vaccine to improve the pentenyl)cyclopentaneacetic acid], and cis-jasmone[3 response to the vaccine, i.e., by inducing a greater response methyl-2-(2-pentenyl)-2-cyclopenten-1-one], 3 ,3' through inducing apoptosis. dihexyloxacarbocyanine iodide, sodium stibogluconate, 20 In one embodiment, a DIM-related indole, optionally With extracts of Yucca schidigera, and curcumin. Apoptosis pro an antiprotoZoal agent, is administered in conjunction With an moting chemotherapeutics include, but are not limited to, anti-protoZoal vaccine, for example, a vaccine Which contains PyrroloquinaZolinediamine, Novobiocin, quercetin, attenuated protoZoal organisms, typically inactivated by irra cyclosporine, dihydrobetulinic acid, campothectins, espe diation and/ or chemical processing. Promotion of more e?i cially topotecan, irinotecan, SN38 (the active metabolite of 25 cient apoptosis of host cells infected With attenuated protoZoa irinotecan), bor‘teZimib, etoposide, quinones including salvi and more effective interaction of protoZoal antigens With host cine, and anthracyclines including doxorubicin, daunorubi immune cells is believed to result from the combined use of cin, 4'-epirubicin, idarlibicin, and deoxydoxorubicin. DIM-related indole With a variety of vaccines. AntiprotoZoal natural products include, but are not limited to, teas and extracts made from Arlemisia annua, teas and 30 4.3 Prevention and Treatment of ProtoZoal Parasite extracts made from Curcuma domeslica, extracts from garlic Infections Which include allicin and other thiosul?nates, root extracts of Uvaria chamae (Annonaceae) and Hippocralea Africana In certain embodiments, the present invention provides for (Hippocrateaceae), and root extracts of Homalium leleslui. the prevention and treatment of protoZoal parasite infections Also useful are extracts of Brasilian plants With demonstrated 35 Which include active infections and asymptomatic chronic anti-malarial activity, including Vernonia brasiliana and infections. Currently, this spectrum of protoZoal parasite Acanlhospermum auslrale (Carvalho, 1991, BraZ J Med Biol infections lacks adequate and effective therapy due to failure Res. 24(11):1113-23, and Botsaris AS. Plants used tradition of previous approaches to selectively eliminate protoZoal ally to treat malaria in BraZil: the archives of Flora Medicinal. parasite infected cells and tissues and to target the anti-apo J Ethnobiol Ethnomed. 2007 May 1; 3:18). 40 ptotic cell signaling pathWays activated by protoZoal para Preferred agents for combined use With DIM-related sites. Ideally, use of DIM-related indoles Will alloW safer and indoles include artemisinin extracts and related drugs, cur more effective treatments of protoZoal infections during preg cumin and curcumin-related drugs, and other antiprotoZoal nancy in immune compromised patients. Treatment of the agents With short metabolic half lives, preferably With a half various protoZoal parasitic diseases varies according to the life similar to DIM (i.e., approximately 2-7 hours). Inuse With 45 parasite type and setting for treatment. DIM-related indoles preferred antimalarials Will be metaboli 4.3.1 General Principles of Treatment for ProtoZoal Para cally cleared Within 12 hours folloWing an oral dose and not sites Using DIM-Related Indoles increase activity of drug metaboliZing cytochrome enZymes DIM-related indoles are used for the treatment and preven (Giao et al., 2001, Clin Pharmacokinet. 40(5):343-73). Addi tion of protoZoal parasite infections in formulations most tional useful natural product preparations are described in the 50 suitable for the disease entity, disease severity, and setting of literature (Willcox et al., 2004, BMJ. 329(7475):1156-9). treatment. An objective of the invention is to eliminate or 4.2.2 Differentiation Promoting Agents shorten the need for parenteral therapy of protoZoal diseases In an additional embodiment, the combination of a DIM and, in doing so, reduce the need for hospitalization. When related indole and a knoWn antiprotoZoal agent is adminis appropriate DIM is used in combination With other desirable tered in conjunction With differentiation promoting agents 55 antiprotoZoal agents to reduce the chance of selecting for Which help protoZoa infected cells develop into more com resistance to DIM-related indole activity. Desirable antipro pletely differentiated and more therapeutically sensitive cells. toZoal agents for combined use possess one or more of the Differentiation promoting agents include Vitamin-D, Vita folloWing important characteristics: safety, stability, chemi min-D derivatives, Vitamin-A (retinoids), retinoid deriva cal compatability in formulations, tolerability, effectiveness, tives, and granulocyte/macrophage colony stimulating fac 60 oral or intravenous dosage form, short elimination half-time, tors including recombinant human Filgrastim and independent mode of action, synergistic activity in vivo. Ide Sargramostim. ally, agents for combination Will have loW cost and alloW a 4.2.3 Immune Potentiating Agents short duration of use in order to achieve durable treatment The DIM-related indoles can also be used in combination response, and be appropriate for both adult and pediatric use. With non-DIM related indole immune potentiating agents. 65 Also important in combined uses and products is compat Immune potentiating agents useful in the methods of the ibility With use in conjunction With antiprotoZoal vaccines, present invention include Aloe vera extracts, mushroom including those Which may be developed in the future. Desir US 8,586,621 B2 21 22 able antiprotoZoal agents for combined use include com rectal DIM-related indole, preferably in a combined supposi pounds Which like DIM-related indoles support selective apo tory With Artemisinin derivative. Those patients able to take ptosis of parasite infected cells. Examples include, but are not oral ?uids simultaneously begin oral DIM-related indole limited to, artemisinin derivatives, atovaquone, chloroquine, preparation as a suspension or in capsules or tablets. Those iodoquinol (diiodohydroxyquin), clioquinol (iodochlorhy subjects With diminished level of consciousness are provided droxyquin), miltefosine, sodium stibogluconate, and cur parenteral hydration ?uids. When available, oxygen by mask cumin. is added to potentiate the anti-parasitic activity of the DIM Also desirable for use in combination With DIM-related related indole and additional anti-protoZoal drug. Repeat indoles, With or Without vaccines, are selected antibiotic or blood smear examination is performed after 3 days to estab chemotherapeutic agents Which promote apoptosis. Desir lish PCT. Oral DIM-related indole therapy is generally given able agents inhibit proteo somal function, induce endoplasmic With a second antimalarial drug, preferably an artemisinin or reticulum stress, inhibit topoisomerase enzymes, inhibit artemisinin derivative, in combination With lumefantrine, vacuolar-H+-ATPase, and/or inhibit famesyl transferase me?oquine, sulfadoxine-pyrimethamine, or amodiaquine in enZymes. Preferred chemotherapeutic agents for use in com chloriquine resistant areas. For use in chloroquine sensitive bination With DIM-related indoles for protoZoal parasites are areas, DIM-related indole therapy is generally given With ?uoroquinolones, enoxacin, cipro?oxacin, Novobiocin, artimisinin and/ or chloroquine. Optimal treatment duration is cyclosporine, luteolin, butoconaZole, sodium butyrate (So from 3 to 14 days. A repeat blood smear for parisitemia is dium butanoate), phenylbutyrate, curcumin, evodiamine, again performed at 14 days and again at 28 days. dihydrobetulinic acid, campothecins, especially topotecan, 4.3.2.3 Use With Anti-Malaria Vaccines irinotecan, borteZimib, etoposide, quinones including salvi 20 FolloWing the administration of a preventive malaria vac cine, and the anthracyclines including doxorubicin, daunoru cine, typically a sporoZoite-based vaccine, oral DIM-related bicin, 4'-epirubicin, idarubicin, and deoxydoxorubicin. indole therapy is initiated 24-96 hrs folloWing receipt of the Desirable antiprotoZoal agents for combination With DIM vaccine. Optimally, the DIM-related indole composition is related indoles can also be chosen to be appropriate for out given as an oral suspension or tablet in combination With one of-hospital, rural use settings. Such agents are intended for 25 or more antimalarial drugs selected from artemisinin deriva distribution and use as nutraceutical products Without pre tives, primaquine, atovaquone, proguanil, or pryimentamine scription or medical supervision. The ingredients need to be sulfadoxine. safe, stable Without refrigeration, and simple to use. Ideally, 4.3.2.4 Prophylactic Use by Non-Immune Individuals. nutraceutical products Will be compatible for use along With In anticipation of exposure to malarial parasites during other natural product , especially tea breWed 30 travel or residence in an endemic area, DIM-related indole fromAlemesia annua, the source of artemisinin. Evodiamine, therapy is initiated on arrival, continued daily during expo an indole alkaloid component extracted from the fruit of sure to risk and further continued 1-2 Weeks after the period of Evodiae Fuctus (Evodia rutaecarpa Benth.) is an alternative exposure. Alternatively, the DIM-related therapy is taken in natural product for use With DIM-related indoles. Additional, combination With Chloroquine phosphate (Aralen) 500 mg DIM-related indole compatible herbal medicines and appro 35 taken once Weekly or in suggested pediatric doses beginning priate use is described in the literature (Willcox et al., 2004, one Week prior to exposure and continuing until 2 Weeks after BMJ 329(7475):1156-9). exposure in chloroquine sensitive areas. Alternatively, in Dosages and treatment regimens for antimalarial agents chloroquine resistant areas, the oral DIM-related indole are taught in Goodman & Gilman’ s The Pharmacologic Basis therapy is taken in conjunction With Atovaqone-proguanin of Therapeutics, Goodman et al. [ed], 9th edition (Jan. 15, 40 (Malarone) in standard doses, With Me?oquine hydro 1996) McGraW Hill Text. cholorid (Lariam) in standard doses, With Doxycycline 4.3.2 Use of DIM-Related Indoles With AntiprotoZoal hyclate in standard doses, or With Primaquine phosphate in Agents for Malaria standard doses. At the option of a supervising physician, the 4.3.2.1 Severe Malaria dose of the non-DIM agents can be reduced during the period In severe Plasmodium falciparum infection With neuro 45 of prophylaxis When using DIM-related indole therapy. Pref logical features, termed cerebral malaria (CM), especially erably, the DIM-related indole is combined in the same tablet impaired consciousness, use of DIM-related indoles is by or capsule With primaquine phosphate. DIM-related indole is parenteral route or rectal suppositories. Intravenous DIM combined With an Artemisinin derivative, and optionally related indole is generally administered in rotation With additional curcumin for use during pregnancy. parenteral quinine or an artemisinin-based drug (e.g., dihy 50 4.3.3 Combined Use of DIM-Related Indoles With Anti droartemisinin, artemether, or artesunate). The rate of intra protoZoal Agents for Trypanosomiasis venous administration of DIM-related indole and additional In treating advanced African trypanosomiasis during the anti-malarial agent(s) is based on renal function, hepatic late menigoencephalitic stage an intravenous suspension or function and overall condition of the patient. This is in addi emulsion of DIM-related indole is alternated With intrave tion to parenteral ?uids, blood exchange transfusion, osmotic 55 nous melarsoprol. The combined use is intended to alloW a diuretics and correction of hypoglycemia, acidosis and hypo reduction of the melarsoprol dose from the typical 2.0-3.6 volemia. Oxygen by mask or mechanical ventilation is added mg/kg per day dose of melarsoprol given for 3-4 days once to potentiate the anti-parasitic activity of the DIM-related Weekly for 3 consecutive Weeks. Alternatively, loWer doses of indole and additional anti-protoZoal drug. Administration of melarsoprol from 1-2 mg/kg per day are given With intrave oxygen therapy is utiliZed to support the apoptotic mecha 60 nous DIM-related indole for 10 continuous days. For infec nisms of action of DIM-related indoles. tion due to T brucei gambiense, intravenous or oral DIM 4.3.2.2 Moderate-Severe Malaria related indole is used in conjunction With intramuscular or Moderately severe malaria is typically characteriZed by intravenous Pentamidine at standard doses for a period of 10 fever and lethargy. When possible, blood tests for parisitemia days. For infection due to T brucei rhodensiense, intravenous should be performed to establish the diagnosis and be 65 or oral DIM-related indole is used in conjunction With intra repeated to document the parasite clearance time (PCT). venous Suranim at standard doses for a period of 10 days. According to the present invention, treatment is begun With Alternatively, in severe cases of African trypanosomiasis, US 8,586,621 B2 23 24 intravenous DIM-related indole is administered in conjunc 4.3.5.1 Use of DIM-Related Indoles With AntiprotoZoal tion With intravenous campothecin derivative such as topo Agents for Toxoplamosis tectan in standard doses for 10 or more days. Treatment of toxoplasmic encephalitis consists of admin In treating American trypanosomiasis (Chaga’s Diseasae) istration of oral DIM-related indoles using a dose range of due to T Cruzi, intravenous or oral DIM-related indole is used 50-200 or 50-300 mg tWice daily, alone or in conjunction With in conjunction With intravenous Suranim at standard doses for pyrimethamine and sulfadiaZine along With folinic acid using a period of 10 days for severe disease. Alternatively, oral established protocols in adults. Children use DIM-related DIM-related indole is administered in conjunction With oral indole as a pediatric suspension providing 25-150 or 25-300 nifurtimox and/or oral bensnidaZole. Alternatively, DIM-re mg tWice daily, alone or in conjunction With a suspension of lated indole is formulated With nifurtimox or bensnidaZole in pyrimethamine and sulfadiaZine along With folinic acid using the same ?avored oral suspension or pill to improve compli established pediatric protocols. Alternatively, capsules or pediatric suspensions containing a combination of DIM, ance and ease of use during chronic treatment. Artemisinin, curcumin, and piperine tWice daily can be uti Alternatively, intravenous DIM-related indole is adminis liZed. DIM-related indole at adult doses alone or With Spira tered in conjunction With intravenous campothectin deriva mycin can be used during pregnancy. tive such as topotectan in standard doses for 10 or more days 4.3.6 Combined Use of DIM-Related Indoles With Anti in severe cases of American trypanosomiasis. ProtoZoal Agents for Trichomonal Disease. 4.3.4 Combined Use of DIM-Related Indoles With Anti Trichomonal vulvo-vaginitis in Women and urethritis in protoZoal Agents for Leishmaniasis men is treated according to the present invention by admin For the treatment of Leishmaniasis, DIM-related indole 20 istering a DIM-related indole in addition to standard doses of therapy is combined With administration of Pentavalent anti oral metronidaZole (Cudmore S L, Delgaty K L, HayWard mony compounds for a more effective therapy. Typically oral McClelland S F, Petrin D P, Garber G E. Treatment of infec or intravenous DIM-related indole is administered in con tions caused by metronidaZole-resistant Trichomonas vagi junction With sodium stibogluconate given at a dose of 20 nalis. Clin Microbiol Rev. 2004 October; 17(4):783-93). This mg/kg/day or less for a period of 28 days to treat visceral 25 can be accomplished by adding DIM, preferably in an absorp leishmaniasis. Alternatively, intravenous DIM-related indole tion-enhanced delivery system (US. Pat. No. 6,086,915), and is administered in conjunction With intravenous campothec administering 100-200 mg orally once or tWice daily for 1-2 tin derivative such as topotectan in standard doses for 10 or Weeks. Alternatively, DIM is formulated as a vaginal cream more days in severe cases of visceral leishmaniasis. For cuta alone or in combination With artesunate for topical adminis neous disease the duration of combined treatment is reduced 30 tration in infected Women. Ideally the vaginal cream is for to 20 days. Optionally, a transderrnal preparation of DIM mulated for sustained delivery using site-directed formula related indoles is added to the orally administered DIM tion technology to produce unit-dose vaginal creams Which related indole and applied topically in cutaneous disease. are retained on the mucosa. Preferred embodiments utiliZe Alternatively, intravenous DIM related indole is administered formulation techniques described in US. Pat. Nos. 6,899, in combination With liposomal amphotericin B Where resis 35 890, 6,214,379, 5,730,997, 5,554,380, 5,445,829, 5,266,329, tance to antimony-based drugs is identi?ed. When possible, and US Patent Applications 20030180366 and 20070154516, oral therapy for cutaneous disease consists of DIM-related 20070224226, 20060140990, and 20040062802. indole given With oral miltefo sine. This combination permits Optionally, the DIM-related indole (50-500 mg) is com a reduction in the standard dose of miltefosine from 100 bined With artemisinin or a derivative such as artesunate, mg/kg/day. In addition, a DIM-related indole can be admin 40 providing 100-300 mg of artesunate per unit dosage. The istered both orally and topically for use in combination With combination cream is used every 1 -3 days in conjunction With paromomysin and/ or additional gentamicin. oral metronidiZole or tinidaZole at established doses. Alter In a alternative preventive approach, a DIM-related indole natively, the DIM-related indole is combined With Butocona is administered orally or parenterally With miltefosine and/or Zole (75-200 mg) or Clindamycin (75-200 mg), With optional a artimesinin derivative in a veterinary formulation formu 45 addition of artemisinin, or artimisinin derivative, to provide a lated as a supplement for dogs or other domestic animals. It treatment for combined protoZoal-bacterial or protoZoal-fun can be given in regional programs to reduce transmission gal infections. In a further embodiment, the DIM-related from dogs or other domestic animals Which serve as a reser indole (50-500 mg) is combined With extracts incorporating voir for the parasite. the active ingredient from the fruit pericarp of Sapindus 4.3.5 Use of DIM-Related Indoles With AntiprotoZoal 50 miukorossi for a topical preparation With both anti-tri Agents for ProtoZoal Diarrheal Disease Due to Crytpospo chomonal and spermicidal activity. ridia or Other Coccidial Protozoa 4.3.6.1 Combined Use of DIM-Related Indoles With Anti DIM related indole therapy for Coccidal diarrhea caused ProtoZoal Agents and Antibiotics for Mixed Vaginal Infec typically by Cryplosporidium part/um, C. hominis, or tions. Cyclospora cayelanensis is based on symptoms and identi? 55 DIM related indoles can be formulated in topical emul cation of the protoZoal oocytes in stool analysis. In adults, sions, some With sustained mucosal retention and release of oral DIM-related indoles are typically employed for treat active agents for use in mixed vaginal infection. These uses ment using a dose range of 50-200 or 50-400 mg tWice daily, include DIM related indoles in combination use With clinca alone or in conjunction With NitaZoxanide 500 mg tWice daily mycin for bacterialiyeast infections, or alternatively, com in adults. Children use DIM-related indole as a pediatric 60 bined With both clindamycin and artemisinin derivative for suspension providing 25-150 or 25-300 mg tWice daily, alone bacterial-yeast infections. DIM related indoles can be used or in conjunction With a suspension of NitaZoxanide provid With artemisinin derivatives for combined trichomonal-yeast ing 200 mg tWice daily. Alternatively, capsules containing a infections, combined With butoconaZole for combined tri combination of DIM, Artemisinin, and curcumin tWice daily chomonal-yeast infections or alternatively, combined With can be utiliZed Without NitaZoxanide. These capsules are also 65 butoconaZole and artemisinin derivatives for combined tri useful in treating Microsporidial infections caused by E mero chomonal-yeast infections. Preferred embodiments utiliZe cylozoon species in immunocompromiZed patients. formulation techniques described in US. Pat. Nos. 6,899, US 8,586,621 B2 25 26 890, 6,214,379, 5,730,997, 5,554,380, 5,445,829, 5,266,329, sion or emulsion to deliver 2-15 mg/kg per dose every 8 to 12 and US PatentApplications 20030180366 and 20070154516, hours. DIM may be administered With quinine in malaria, 20070224226, 20060140990, and 20040062802. Optionally, With e?omithine, pentamidine, melarsoprol, nifurtimox, or DIM related indoles can be used in combination With other benZnidaZole, for tryanosomiasis, and With pentavalent anti useful antiprotoZoal agents including metronidaZole, or in mony, amphotericin B, or miltefosine in Leishmaniasis. combination With useful anti-fungal agents, including aZole For life threatening conditions, intravenous DIM-related drugs, particularly butoconaZole. In addition, natural prod indoles Would be administered With or Without DIM supposi ucts drugs or extracts of medicinal plants can be used in tories and/or additional aerosoliZed DIM. Oxygen therapy is combination With DIM related indoles and optional anti added to potentiate parasite clearance using DIM-related protoZoal, anti-bacterial, or anti-fungal agent. Preferred natu indoles. ral products for combined use include evodiamine, curcumin, extracts of Sapindus mukorossi, and sodium butyrate. Addi 4.4 Administration and Dosage tionally, DIM related indoles, and optional additional anti protoZoal agent, can be combined With topically active sper macides for preparations With both microbicidal and In certain embodiments, certain combinations of DIM contraceptive activities. related indoles, e.g., DIM, and one or more knoWn antipro 4.3.7 Use of DIM-Related Indoles for Intestinal ProtoZoal toZal agents in parenteral delivery systems, oral delivery sys Infections in Livestock tems, rectal suppositories, vaginal creams, or by In certain embodiments of the invention, formulations simultaneous delivery by multiple routes provides therapeu include combined use of DIM-related indoles With antipro 20 tic e?icacy are believed to provide more than the additive toZoal agents to treat intestinal protoZal infections in livestock e?icacy of each agent used alone at maximal dose. Therefore, due to coccidial or neospora protoZoal parasites. In particular, methods involving combined use of a DIM-related indole and for additives to cattle feed to prevent crytosporidal infection a knoWn antiprotoZal agent at less than their maximal doses associated abortion during breeding, preferred feed additives increase both the safety and e?icacy of DIM-related indoles utiliZe, for example, DIM-related indoles, genistein, extracts 25 and antiprotoZal agents in selected protoZoal infections. or derivatives of Artemisia annua, and extracts of Curcuma Improved ef?cacy results in a shorter duration of required domeslica. Also useful in combination With DIM-related therapy than With individual agents used alone. Combined indoles are extracts of Yucca schidigera. use can alloW a reduction in dose or shortening of the period 4.3.8 Use of DIM-Related Indoles in Combination With of high dose treatment. Combined use can improve the long Artemisinin-Based Drugs. 30 term therapeutic result With a loWer rate of recrudescence Typically in human use a tWice daily oral dose of 50-250 or With reneWed appearance and groWth of surviving parasites. 50-600 mg/day (1-3 or 1-6 mg/kg/day) of a DIM-related Combined use With loWered dose and duration of use can indole in a suitable formulation is taken along With a tWice minimiZe toxicity. daily oral dose of 25-1000 mg (0.5-10 mg/kg/day) of dihy In methods involving the oral use of one or more DIM droartemisinin, artesunate or other artemisinin-derived drug 35 related indoles, e.g., DIM, and one or more knoWn antipro in a suitable formulation. toZal agents, the oral delivery of indole is facilitated and In a preferred embodiment, a tWice daily oral dose of accomplished according to formulations and methods 50-550 mg/day (1-3 mg/kg/day) of DIM-related indole in a described in US. Pat. No. 6,086,915, incorporated by refer suitable formulation is taken along With a tWice daily oral ence herein in its entirety. In one embodiment, DIM, or a dose of 25-1000 mg (0.5-10 mg/kg/day) of dihydroartemisi 40 DIM-related indole, is processed With phosphatidyl choline. nin, artesunate, or other artemisinin-derived drug in a suitable Alternatively, oral and rectal bioavailability of DIM-related formulation, together With curcumin at a tWice daily oral dose indoles are improved using other means including particle of 25-1000 mg (0.5-10 mg/kg/day) and piperine to aid siZe reduction, complexation With phosphatidylcholine, and absorption. Typically 20-150 mg of DIM related indole is formation into rapidly dissolving particles and nanoparticles. combined With 25-1500 mg (0.5-10 mg/kg/day) Artemesinin 45 The treatment of protoZoal disease With an oral DIM-re extract and 25-1500 mg (0.5-10 mg/kg/day) of curcumin and lated indole, e.g., DIM, is facilitated by oral, sublingual, 20 mg of piperine 20 mg. Preferably the extract of piperine is intravenous, rectal, vaginal, transdermal, and intra-lesional called Bioperine® (an extract from the fruit of Piper nigrum application of DIM-related indoles in speci?c relative doses L (black pepper) or Piper longum L (long pepper) containing With simultaneous administration of a knoWn antiprotaZoal 95 percent piperine; Sabinsa Corporation, PiscataWay, N.J.). 50 agent. These therapies include production of tinctures, lipo The DIM-related indole, artemisinin-related drug, curcumin somes, creams, or rectal/vaginal suppositories, emulsions for and piperine are preferably contained in the same capsule or intravenous use, and injectable suspensions to deliver syner tablet to facilitate combined use. gistic amounts of these agents. Injectable formulations In a second preferred embodiment, the DIM-related indole include cyclodextrin complexed DIM-related indoles and and artemisinin derivative (preferably artesunate) is forrnu 55 liposome encapsulated DIM-related indoles. lated using site-directed formulation technology to produce For oral use, DIM is used, preferably formulated for unit-dose vaginal or rectal suppositories/creams Which are enhanced absorption in a daily dose of 0.5-12 mg/kg per day. retained on the mucosa for sustained delivery. Related, Oral DIM is optionally combined With other oral agents for optional embodiments utiliZes formulation techniques malaria, trypanosomiasis, leishmaniasis, crytosporidiosis, described in US. Pat. Nos. 6,899,890, 6,214,379, 5,730,997, 60 and toxoplasmosis using standard doses of the additional 5,554,380, 5,445,829, 5,266,329, and US PatentApplications agents. 20030180366 and 20070154516, 20070224226, For intravenous use, DIM is used, preferably formulated as 20060140990, 20040062802, and 20020044961. an intravenous suspension or emulsion, in a daily dose of For severe protoZoal disease as seen With cerebral malaria, 0.5-15 mg/kg per day. Intravenous DIM is optionally com African sleeping sickness, acute Chaga’s disease, and vis 65 bined With other intravenous and oral agents for malaria, ceral Leismaniasis, the DIM-related indole is preferably give trypanosomiasis, leishmaniasis, crytosporidiosis, and toxo intravenously. DIM is given in a suitable intravenous suspen plasmosis using standard doses of the additional agents. US 8,586,621 B2 27 28 For rectal use, DIM is preferably formulated for enhanced 1. Dry particle complex for oral use rectal absorption in suppositories in a daily dose of 05-12 2. Intravenous emulsion mg/kg per day. DIM is optionally combined With other rectal 3. Parenteral emulsion agents for malaria (artesunate, artemether), trypanosomiasis IV. Nanoparticle-Based Dispersions (e?omithine, ni?lrtimox), leishmaniasis (miltefosine), Examples of manufacturing techniques are described in crytosporidiosis (artesunate, artemether), and toxoplasmosis US. Pat. Nos. 6,288,040; 6,165,988; 6,117,454; and US. (pyrimethamine and sulfadiaZine) using standard doses of the Patent Application Publication No. 20030032601; each of additional agents. Which is incorporated by reference in its entirety. Using nano For vaginal or rectal use the DIM-related indole is formu particle production techniques to incorporate DIM-related lated using site-directed formulation technology to produce indoles the folloWing ?nal formulations are produced: unit-dose vaginal or rectal suppositories/creams Which are 1. Dry particle complex for oral use. retained on the mucosa for sustained delivery. Preferred 2. Intravenous emulsion embodiments utiliZe formulation techniques described in 3. Parenteral emulsion US. Pat. Nos. 6,899,890, 6,214,379, 5,730,997, 5,554,380, V. Liposome Based Formulations 5,445,829, 5,266,329, and US Patent Applications 5 Examples of manufacturing techniques are described in 20030180366 and 20070154516, 20070224226, US. Pat. Nos. 4,906,476; 5,006,343; and US. Patent Appli 20060140990, 20040062802, and 20020044961. Optionally, cation Publication No. 20030108597. Using liposome pro the DIM-related indole is combined With artemisinin deriva duction techniques to incorporate DIM-related indoles the tive (e.g., artesunate), butoconaZole, tinidaZole, niridaZole, folloWing ?nal formulations are produced: nitaZoxanide, or miltefosine, 20 1. Dry particle complex for oral use 2. Intravenous emulsion 4.5 Pharmaceutical/Nutraceutical Compositions 3. Parenteral emulsion 4.5.1 Oral Combined Products Phar'maceutical/Nutraceutical Dosage Forms for DIM-re Combined formulations for oral use include DIM-related lated indoles: Multi-application DIM-related indole contain 25 indole, optionally formulated for enhanced absorption, com ing particles are manufactured by various techniques includ bined With one or more additional anti-protoZoal compounds ing spray drying, spray cooling, selective precipitation, and optionally include additional absorption enhancers. crystallization and other particle forming methods. The Examples of preferred antiprotoZoal compounds include arte resulting particles are used in the manufacture of the folloW misinin; dihydroar‘temisinin; artemethe; artesunate; atova ing dosage forms, some of Which are described in US. Pat. 30 quone; diaminopyrimidines, especially amodiaquine, No. 6,086,915, incorporated by reference herein in its amphotericin, clindamycin, e?omithine, fumagillin; the entirety. 8-hydroxyquinolines, chloroquine, me?oquine, halofantrine, I. Spray Dried Microencapsulated Solid Dispersions lumefantrine, geldanamycin, iodoquinol (diiodohydrox 1. TPGS/phosphospholipid spray-dried particles. Produc yquin) and clioquinol (iodochlorhydroxyquin); the 2-ni tion of absorption-enhanced DIM-related indole particle 35 troimidaZoles including EtanidaZole and BenZnidaZole; formation is provided in US. Pat. No. 6,086,915. doxycycline; melarsoprol; metronidaZole; miltefosine; nifur 2. Liquid emulsions using TPGS/phosphospholipid spray timox; nitaZoxanide; paromomycin; pentamindine; sodium dried particles. Production of emulsions for oral use stibogluconate and related antimonials; suramin; utiliZes absorption-enhanced DIM-related indole par pyrimethamine; proguanil (chloroguanide); spiramycin; sul ticle formation is provided in the US. Pat. No. 6,086, 40 fadoxine; sulfonamides including trimetoprim; sulfones; and 915. tetracyclines; quinine derived from the bark of the South 3. Flavored DIM granules for oral use (Chocolate, Orange American chinchona tree; 6-gingerol and/or 6-paradol (Surh “sprinkles”). Production of ?avored granules for oral et al., 1999, J Environ Pathol Toxicol Oncol. 18(2): 131-9); use utiliZes absorption-enhanced DIM-related indole and selected ?avonoids and derivatives (Tasdemir et al., 2006, particles (DIM/TPGS) as provided in US. Pat. No. 45 Antimicrob Agents Chemother. 50(4):1352-64). Other anti 6,086,915. Production steps include dry mixing DIM/ protoZoal compounds include, but are not limited to, cur TPGS particles With maltodextrin granules, addition of cumin, an extract of Curcuma domeslica, leutiolin, selenium ?avoring particles and granulation using a standard ?uid compounds, especially methylselenic acid, resveratrol, bed granulator. including an extract of Polygonium cuspidalum, silibinin, an II. Spray Dried Polymer Based Solid Dispersions 50 extract of Silybum marianum, apigenin, deguelin, extracted Production techniques for DIM-related indoles may utiliZe from various plant sources including Munduelea sericea, those described in US. PatentApplication No. 20030072801, Evodiamine, ursolic acid, Andrographolide, Dehydro-An entitled “Pharmaceutical compositions comprising drug and drographolide, Deoxy-Andrographolide, Brassinin, Caffeic concentration-enhancing polymers,” herein incorporated by acid, Capsanthin, Capsaincin, Chelerythrine Chloride, Cro reference in its entirety. In particular production involves the 55 molyn sodium, Allyl Disul?de, Diallyl disul?de, Diallyl sul folloWing dissolution enhancing polymers, used With and ?de, Diallyl trisul?de, DibenZoylmethane, Ebulin 1, Ellagic Without lipid stabiliZers: acid, Ferulic acid, 18[3-Glycyrrhetinic Acid, GlycyrrhiZic 1. Polymer included: Hydroxy Propyl Methylcellulose acid ammonium salt trihydrate, Honokiol, 5-Hydroxy-L 2. Polymer: Hydroxy Propyl Cellulose tryptophan, Hypericin, Hypocrellin A, Idebenone, luteolin, III. Cyclodextrin Based Formulations 60 D-Limonene, Limonin, Limonin Glucoside, DL-ot-Lipoic Examples of manufacturing techniques are described in acid, Melatonin, Perillyl Alcohol, Phenylbutyrate, Phenyl US. Pat. No. 4,877,778 and US. Patent Applications Nos. ethyl 3-methylcaffeate, Phenylethy 14-methylcaffeate, Phe 20040053888; 20030073665; and 20020068720, each of nyl isothiocyanate, Phytic Acid, Rosmarinic acid, Rutae Which is herein incorporated by reference in its entirety. carpine, , L-Threonine, , aspirin, Using cyclodextrin loading production techniques to incor 65 salycylamide. Absorption-enhancing agents can be addition porate DIM-related indoles the folloWing ?nal formulations ally added including, but not limited to, Vitamin-E polyeth are produced: yleneglycol succinate (TPGS), piperine, limonine, D-Li US 8,586,621 B2 29 30 monene, and/ or polyethyleneglycol. In addition, orally active Candidal infection, and Crytosporidial infection. For vaginal immune potentiating agents can be utilized in addition to or rectal use the DIM-related indole is formulated using site anti-protoZoal agents including Vitamin K3, N-Acetyl-L directed formulation technology to produce unit-do se vaginal Cysteine, Zinc citrate, or Zinc gluconate. or rectal suppositories/creams Which are retained on the The DIM, or a DIM-related indole, together With one or mucosa for sustained delivery. One related embodiment uti more antiprotoZoal compounds and optionally, an antiproto liZes formulation techniques described in US. Pat. Nos. Zoal plant extract can also be added to selected foods as 6,899,890, 5,554,380, 5,266,329, and US PatentApplications forti?ed, “functional” foods. Forti?ed foods include “medici 20030180366 and 20070154516. Optionally, the DIM-re nal foods” Which require use under a doctor’s care and “func lated indole is combined With artemisinin derivative (eg, arte tional foods” available to consumers as unregulated special sunate), butoconaZole, clindamycin, tinidaZole, niridaZole, iZed foods. Such uses in forti?ed or “functional” foods nitaZoxanide, or miltefosine, typically apply to Food Bars, Drink Mixes, Vegetable Juices, 4.5.3 Hepatic Targeted Formulations Pasta Mixes, Dry Cereal, Meal Replacement PoWders, Tea Hepatic targeting includes intravenous emulsions Which mixes, and Baked Goods. Such uses require specialiZed pro are concentrated in liver tissue With or Without lipids and, duction With the dose of DIM in accordance With principles of optionally including anti-protoZoa-speci?c antibodies. Con Generally RegardedAs Safe (GRAS) food ingredients. These centration in hepatic tissue provides an advantage in treating typically include drink mixes, meal replacement poWders, the hepatic stage of malaria. food bars, and candies. Formulations include DIM, or a DIM 1. Phospholipid complexed intravenous emulsions related indole, and one or more of the folloWing antiprotoZoal 2. Cyclodextrin-based intravenous emulsions compounds: artemisinin, genistein, curcumin, and resvera 20 3. Intravenous suspension complexed With Anti-protoZoal trol. The compositions of DIM, and DIM-related indoles, of monoclonal antibodies the present invention are also utiliZed as added ingredients to 4.5.4 Leukocyte Targeted Formulations forti?ed foods to facilitate convenient and regular consump Leukocyte targeting includes intravenous emulsions Which tion to prevent or control chronic protoZoal parasite infection. are concentrated in leukocytes With or Without lipids and AntiprotoZoal forti?ed foods With DIM are used in periodic 25 Anti-protoZoa-speci?c antibodies. Concentration in leuko “cleansing” programs, Where special diets and intermittent cytes provides an advantage in treating the leukocyte stage of fasting is use to reduce intestinal parasite infection. In such trypanosomal and leishmanial infections. applications DIM is added to food products or mixes alone or 1. Phospholipid complexed intravenous emulsions in combination With selected antiprotoZoal compounds, par 2. Cyclodextrin-based intravenous emulsions ticularly artemisinin, curcumin, and iso?avones like 30 3. Intravenous suspension complexed With Anti-ProtoZoal genistein. monoclonal antibodies Food Bar Products are produced according to the present 4.5.5 DIM Tinctures and Suspensions invention according to knoWn manufacturing and baking Tincture preparation alloWs a simple absorbable liquid for practices. Detailed of food bar composition and manufactur mulation of DIM-related indole to be prepared for use in a ing techniques useful With DIM, DIM-Related Indoles, and 35 rural setting. A mixture of DIM-related indole is manufac DIM combined With selected antiprotoZoal compounds are tured With optional, additional anti-protoZoal compound speci?ed in US. Patent Application Publication Nos. using ethanol as a solvent to provide various tincture formu 20030068419 entitled “Food bar compositions” and lations. Methods for making tintures appropriate for anti 20020168448 entitled “Nutritional food bar for sustained protoZoal compounds are described by SWeet in US. Pat. No. energy”. 40 7,033,619, entitled “Method for making herbal extracts using Drink Mix Products are produced according to the present percolation” and methods speci?c for Atemesinin tinctures invention according to knoWn manufacturing practices. described in US. Pat. No. 4,952,603. Detailed drink mix composition and manufacturing tech 1. Oral tinctures prepared With ethanol or hexanol niques useful With DIM, DIM-Related Indoles, and DIM 2. Parenteral tincture prepared With ethanol or hexanol combined With selected antiprotoZoal compounds are speci 45 3. Microcrystalline suspension for oral use ?ed in US. Pat. No. 6,599,553 by Kealey et al., entitled “Dry 4. Parenteral microcrystalline suspension drink mix and chocolate ?avored drink made therefrom”. 5. Sustained release parenteral suspension In preferred embodiments, DIM is incorporated in forti?ed Pharmaceutical compositions according to the present foods, such as drink mixes and food bars, during food pro invention preferably comprise one or more pharmaceutically duction using a particulate form of DIM that is formulated for 50 acceptable carriers and the active constituents, e.g., a DIM enhanced absorption (BioResponse-DIM [BioResponse, related indole alone, or a DIM-related indole and one or more LLC, Boulder, Colo.]). Artemisin is added as a poWdered knoWn antiprotoZal agents. The carrier(s) must be “accept extract of Arlemisia annua (Artemisin [Allergy Research able” in the sense of being compatible With the other ingre Group, Alameda, Calif.]). Genistein is added as a poWdered dients of the composition and not deleterious to the recipient formulation of pure iso?avone (Bonistein [DSM Nutritional 55 thereof. Products]). Typically, the DIM is provided in a dose of 10-75 The term “carrier” refers to a diluent, adjuvant, excipient, mg/ serving (40-300 mg/ serving of BioResponse-DIM). Arte or vehicle With Which the therapeutic is administered. The misinin is provided in a dose of 50-300 mg/ serving. Genistein carriers in the pharmaceutical composition may comprise a is provided in a dose of 25-100 mg/ serving as Bonistein. binder, such as microcrystalline cellulose, polyvinylpyrroli 4.5.2 Mucosally Targeted Formulations 60 done (polyvidone or povidone), gum tragacanth, gelatin, Mucosally targeted formulations includes specialiZed starch, lactose or lactose monohydrate; a disintegrating agent, emulsions of DIM-related indoles, optionally combined With such as alginic acid, maiZe starch and the like; a lubricant or knoWn anti-protoZoal agents Which are retained on and pen surfactant, such as magnesium stearate, or sodium lauryl etrate the targeted mucosa for long periods of time. This sulphate; a glidant, such as colloidal silicon dioxide; a sWeet includes specialiZed formulations for oral, esphogeal, rectal, 65 ening agent, such as sucrose or saccharin; and/or a ?avoring vaginal, and vulvar mucosa. This formulation technology agent, such as peppermint, methyl salicylate, or orange ?a provides a preferred Way of treating Trichomonal infection, voring. US 8,586,621 B2 31 32 It Will be appreciated that the amounts of DIM or other synthetic origin, such as peanut oil, soybean oil, mineral oil, DIM-related indole and/or one or more known antiprotoZal sesame oil and the like. Formulations for parenteral admin agents, required for the treatments disclosed herein Will vary istration also include a lyophiliZed poWder comprising phy according to the route of administration, the severity of the tochemical that is to be reconstituted by dissolving in a phar protoZoal disease, age, and medical history of the subject, the maceutically acceptable carrier that dissolves said galenic formulation of the pharmaceutical composition, etc. phytochemical. Parenteral administration also includes a Preferably, the DIM used in the invention has been pro stable emulsion of DIM designed for intravenous use. Ideally, cessed to enhance bioavailability, as is described in US. Pat. the emulsion prevents the early removal of DIM from the No. 6,086,915, incorporated herein by reference in its circulation due to early uptake by the reticulo-endothelial entirety; hoWever any suitable preparation of pure diindolyl system alloWing maximal cellular concentration of DIM in methane can be used in the methods and compositions of the invention. Optionally, additional absorption enhancing parasite-infected cells or tissue. agents active With DIM-related indole such as grapefruit When the pharmaceutical composition is a capsule, it may extracts or extract of black pepper providing extracts of pip contain a liquid carrier, such as a fatty oil, e.g., cacao butter. erine can be included. Suitable pharmaceutical excipients include starch, glu In general, a suitable (therapeutically effective) amount of cose, lactose, sucrose, gelatin, malt, rice, ?our, chalk, silica Diindolylmethane is preferably administered in an absorp gel, sodium stearate, glycerol monostearate, talc, sodium tion enhancing formulation, as described in US. Pat. No. chloride, dried skim milk, glycerol, propylene, glycol, Water, 6,086,915, at 25-750 mg per day as a suspension of micro ethanol and the like. These compositions can take the form of particles in a starch carrier matrix. Structurally-related, syn 20 thetically-derived, substituted diindolylmethane’s, as solutions, suspensions, emulsion, tablets, pills, capsules, described by long (US. Patent Application Publication No. poWders, sustained-release formulations and the like. The 2004/0043965) are administered according to the present composition can be formulated as a suppository, With tradi invention in an acceptable formulation for oral administration tional binders and carriers such as triglycerides or nerolidol, in a dose of 10-400 mg/day. Preferably, these substituted 25 a sesquiterpene. diindolylmethanes are administered in an absorption-en In yet another embodiment, the therapeutic compound can hanced formulation at a dose of 50 to 250 mg/day. The actu be delivered in a controlled release system. In one embodi ally administered amounts of DIM or a substituted diindolyl ment, a pump may be used (see Langer, supra; Sefton, CRC methane may be decided by a supervising physician. The Crit. Ref. Biomed. Eng. 1987, 14:201; BuchWald et al., Sur DIM-related indole of the invention is preferably adminis 30 gery 1980, 88:507; Saudek et al., N. Engl. J. Med. 1989, tered in combination With one or more knoWn antiprotoZal 321:574). In another embodiment, polymeric materials can agents administered by either oral, rectal, or parenteral routes. be used (see Medical Applications of Controlled Release, Therapeutic formulations include those suitable for Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); parenteral (including intramuscular and intravenous), topical, Controlled Drug Bioavailability, Drug Product Design and oral, vaginal, rectal or intradermal administration. Thus, the 35 pharmaceutical composition may be formulated as tablets, Performance, Smolen and Ball (eds.), Wiley, NeW York pills, syrups, capsules, suppositories, ophthalmic suspension, (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macro formulations for transdermal application, poWders, espe mol. Chem. 1983, 23:61; see also Levy et al., Science 1985, cially lyophiliZedpoWders for reconstitution With a carrier for 228: 1 90; During et al., Ann. Neurol. 1989, 25:35 1; HoWard et intravenous administration, etc. Oral administration for DIM 40 al., J. Neurosurg. 1989, 71:105). is the most preferred route. Other controlled release systems are discussed in the Therapeutic formulations suitable for oral administration, revieW by Langer (1990, Science 249: 1527-1533). e.g., tablets and pills, may be obtained by compression or In one embodiment of the pharmaceutical composition molding, optionally With one or more accessory ingredients. according to the invention, the DIM-related indole and one or Compressed tablets may be prepared by mixing phytochemi 45 cals, and compressing this mixture in a suitable apparatus into more knoWn antiprotoZal agents are comprised as separate tablets having a suitable siZe. Prior to the mixing, the DIM entities. The entities may be administered simultaneously or related indole or one or more antiprotoZoal agents may be sequentially. mixed With a binder, a lubricant, absorption enhancer, an inert The invention also provides a pack or kit comprising one or diluent and/ or a disintegrating agent. 50 more containers ?lled With one or more of the ingredients of In a preferred embodiment, the DIM-related indole is the compositions of the invention. This includes the combi mixed With a binder, such as microcrystalline cellulose, and a nation of capsules for oral use and rectal suppositories. surfactant, such as sodium lauryl sulphate until a homoge Optionally associated With such container(s) can be a notice neous mixture is obtained. Subsequently, another binder, in the form prescribed by a governmental agency regulating such as polyvinylpyrrolidone (polyvidone), is transferred to 55 the manufacture, use or sale of pharmaceuticals or biological the mixture under stirring With a small amount of added products, Which notice re?ects approval by the agency of Water. This mixture is passed through granulating sieves and manufacture, use or sale for human administration. dried by desiccation before compression into tablets in a standard tableting apparatus. A number of references have been cited, the entire disclo A tablet may be coated or uncoated. An uncoated tablet 60 sures of Which are incorporated herein by reference. may be scored. A coated tablet may be coated With sugar, Many modi?cations and variations of this invention can be shellac, ?lm or other enteric coating agents. made Without departing from its spirit and scope, as Will be Therapeutic formulations suitable for parenteral adminis apparent to those skilled in the art. The speci?c embodiments tration include sterile solutions or suspensions of the active described herein are offered by Way of example only, and the constituents. An aqueous or oily carrier may be used. Such 65 invention is to be limited only by the terms of the appended pharmaceutical carriers can be sterile liquids, such as Water claims along With the full scope of equivalents to Which such and oils, including those of petroleum, animal, vegetable or claims are entitled. US 8,586,621 B2 33 34 5. EXAMPLES pensions are manufactured using techniques described by Kulkami et al. in US. Patent application No. 20050136114, 5.1 Example 1 published June 23, 200, and titled, “Taste masked pharma ceutical compositions comprising bitter drug and pH sensi Manufacture of Processed DIM for Enhanced Oral tive polymer”. Bioavailability In a preferred embodiment the DIM-related indole is for mulated into a pediatric suspension in combination With an Preparation of Processed Diindolylmethane is Accom artemisinin derivative, such as artesunate, curcumin and pip plishedAccording to the steps outlined in US. Pat. No. 6,086, erine utiliZing taste masking. 915, herein incorporated by reference in its entirety. Brie?y, this included mixture of about 10-40% by ?nal Weight of 5.4 Example 4 Diindolylmethane With about 10-40% by ?nal Weight of vita min E polyethylene glycol 1000 succinate (Vitamin-E-TPGS, Manufacture of DIM With Artemisinin Derivative in Eastman Chemical), 2-20% by ?nal Weight, phosphatidyl a Suppository for Vaginal or Rectal Administration choline (Phospholipon 50G, Rhone Poulenc) and 15-30% by 5 ?nal Weight hexanol. This mixture is made homogeneous by In a heated vessel, 90 grams cetostearyl alcohol (Alfol mixing. The homogeneous mixture of indoles and other oil 16/ 18, Vista) mixed With 10 cc Grapefruit Oil (Aldrich soluble substituents listed above is added to a solution of Chemical) Was heated to 1000 C. to Which 20 gms of micro modi?ed starch in Water (Cap sul Starch from National Starch, crystalline DIM, 5 or 10 gms of Artesunate (LKT Labs, St. Inc.). The starch component forms from 30-70% of the ?nal 20 Paul, Minn.), Were added With constant mixing to form a hot dry Weight of the product. The Well dispersed ?nal combined slurry. Alternatively, 90 grams cetostearyl alcohol (Alfol mixture is then subjected to spray drying. The resultant prod 16/18,V“1sta) is heated to 1000 C. to Which 10 gms ofmicro uct is a ?ne poWder containing Diindolylmethane contained crystalline DIM is mixed and to Which is added in a second Within the starch particles. vessel 400 gms of IV Novata (Semi-synthetic Glyceride Sup 25 pository Base, Ashland Chemicals) Was Warmed to 400 C. 5.2 Example 2 With constant mixing. The Well mixed slurry from the ?rst vessel Was added With continued mixing to the second vessel. Manufacture of Capsules Containing The homogenized molted suppository material Was formed Diindolylmethane and Optional AntiprotoZoal into suppositories of 2 g each and cooled. Glyceryl monster Agent(s) 30 ate 10-50 g Was added to the molten mixture as needed to increase the ?rmness of the ?nal suppositories. Optionally, Capsules containing 150-300 mg of processed Diindolyl 50-100 mg of artesunate or arthemether is added per supposi methane, as produced according to the steps described in tory for children, and 200-300 mg of artesunate or artemether example 6.1, are made by mixing the processed Diindolyl is added per suppository for adults. methane With microcrystalline cellulose and placing the 35 Other methods of producing suppositories are Well knoW in mixed poWder into opaque gelatin capsules. the art as described in US. Pat. No. 4,164,573 by Galinsky et Capsules containing the combination of about 150 mg of al., issued Aug. 14, 1979 and titled, “Composition and processed DIM (providing 50 mg of DIM) and 200-500 mg of method for making a suppository for introducing a hypogly Artemisinin, and/ or other Artemisinin derivatives are made cemic agent into a mammal”. Use of this method provides a by mixing the processed DIM, and Artemisinin With micro 40 technique to manufacture a mixture of DIM and Artemisinin crystalline cellulose or rice ?our excipient and placing the related compound Which is then evaporated into a semisolid mixed poWder into opaque gelatin capsules. Alternatively, mass and then shaped into suppository. In a preferred product capsules or tablets With about 150 mg of processed DIM format the suppository Would contain 50-100 mg of DIM and providing 50 mg of DIM and 200-500 mg of Artemisinin, or 50-100 mg of artesunate for children, and 100-300 mg of other Artemisinin derivatives, Curcumin 100-200 mg, and 45 DIM and 200-300 mg of artesunate for adults. Optionally, piperine 20 mg are made With microcrystalline cellulose or Vitamin E TPGS (Eastman Chemical) or nerolidol, a sesquit rice ?our excipient and by placing the mixed poWder into erpene, are added to suppository formulations as drug pen opaque gelatin capsules. etration enhancers.

5.3 Example 3 50 5.5 Example 5

Manufacture of Flavored, Pediatric Suspensions of Manufacture of Sustained-Release Combination DIM-Related Indoles Formulas for Vaginal or Rectal Administration

DIM-related indoles are incorporated into pediatric sus 55 Mucosally targeted formulations includes specialiZed pensions manufacture as poWdered mixtures to be re-consti emulsions of DIM-related indoles, optionally combined With tuted With Water prior to use in patients. The suspension knoWn anti-protoZoal agents Which are retained on and pen products have the advantages of long shelf life, stability and etrate the targeted mucosa for long periods of time. This ?avoring for taste masking to improve palatability. includes specialiZed formulations for oral, esphogeal, rectal, A DIM-related indole containing suspension is made using 60 vaginal, and vulvar mucosa. This formulation technology established manufacturing techniques as described in US. provides a preferred Way of treating Trichomonal infection, Pat. No. 6,586,012 byYu et al. issued Jul. 1, 2003, and titled, Candidal infection, and Crytosporidial infection. For vaginal “Taste masked pharmaceutical liquid formulations”. When or rectal use the DIM-related indole is formulated using site reconstituted, the pediatric suspension has a DIM related directed formulation technology to produce unit-do se vaginal indole concentration of 15-30 mg/ml of suspension. Typi 65 or rectal suppositories/creams Which are retained on the cally, 3-10 mg/kg/ dose of DIM related indole is administered mucosa for sustained delivery. One related embodiment uti tWice daily. Alternatively, DIM-related indole pediatric sus liZes formulation techniques described in US. Pat. Nos.